Scavenging reactive oxygen species using tempol in the acute phase of renal ischemia/reperfusion and its effects on kidney oxygenation and nitric oxide levels
Animals
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 guidelines for Institutional and
Animal Care and Use Committees. The study has been carried out in accordance with
the Declaration of Helsinki. The experiments were performed on 24 Sprague-Dawley rats
(Harlan Netherlands BV, Horst, The Netherlands) with a mean?±?SD body weight of 348?±?21 g.
Surgical preparation
All the animals were anesthetized with an intraperitoneal injection of a mixture of
75 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
0.05Â mg/kg atropine-sulfate (Centrafarm Pharmaceuticals BV, Etten-Leur, The Netherlands).
After preparing a tracheotomy, the animals were mechanically ventilated with a FiO
2
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 pCO
2
between 30 and 35Â mmHg and arterial pCO
2
between 35 and 40Â mmHg.
For drug and fluid administration and hemodynamic monitoring, vessels were cannulated
with polyethylene catheters with an outer diameter of 0.9Â mm (Braun, Melsungen, Germany).
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 jugular vein was cannulated
for continuous infusion of Ringer’s lactate (Baxter, Utrecht, The Netherlands) at
a rate of 15Â mL/kg/h and maintenance of anesthesia. The right femoral artery was cannulated
for drawing blood samples and the right femoral vein for fluid resuscitation.
The left kidney was exposed, decapsulated, and immobilized in a Lucite kidney cup
(K. Effenberger, Pfaffingen, Germany) via ~4Â cm incision in the left flank in each
animal. The renal vessels were carefully separated under preservation of nerves and
the 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, USA)
and connected to a flow meter (T206, Transonic Systems Inc., Ithaca, NY, USA) to continuously
measure renal blood flow (RBF). An estimation of the renal vascular resistance (RVR)
was made as: RVR (dynes.sec.cm
?5
)?=?(MAP/RBF)?×?80. The left ureter was isolated, ligated, and cannulated with a polyethylene
catheter for urine collection.
After the surgical preparation, one optical fiber was placed 1Â mm above the decapsulated
kidney and another optical fiber was placed 1Â mm above the renal vein to measure renal
microvascular and venous oxygenation using phosphorimetry (explained in more detail
below). A small piece of aluminum foil was placed on the dorsal side of the renal
vein to prevent contribution of the underlying tissues to the phosphorescence signal
in the venous pO
2
measurements. Oxyphor G2, a two-layer glutamate dendrimer of tetra-(4-carboxy-phenyl)
benzoporphyrin (Oxygen Enterprises Ltd., Philadelphia, PA, USA), was subsequently
infused (i.e., 6Â mg/kg IV over 5Â min), followed by 30Â 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
After a stabilization period of 30Â min, the animals were randomly divided into four
groups of six: (1) no I/R, no tempol (CTRL); (2) no I/R, but with tempol (TMPL); (3)
I/R without tempol (I/R); and (4) I/R with tempol (I/R?+?TMPL). Ischemia/reperfusion
was induced by 30-min non-destructive clamping of the renal artery. The tempol-treated
animals received 200Â ?mol/kg/h of 4-hydroxy-TEMPO (tempol) intravenously 15Â min prior
to initiation of I/R. Measurements were performed up to 90Â min post-ischemia, and
after the experiments, the kidneys were isolated and renal tissue malondialdehyde
(oxidative stress marker) and nitric oxide levels were measured.
Blood variables
Arterial blood samples (0.5Â ml) were taken from the femoral artery at baseline (BSLN)
and after 15 and 90Â min of reperfusion (R15 and R90, respectively). The blood samples
were replaced by the same volume of Ringer’s lactate. The samples were analyzed for
blood gas values (ABL505 blood gas analyzer; Radiometer, Copenhagen, Denmark), hemoglobin
concentration, and hemoglobin oxygen saturation (OSM3; Radiometer, Copenhagen, Denmark).
Additionally, plasma creatinine and sodium concentrations were determined in all the
samples.
Renal microvascular and venous oxygenation
Microvascular oxygen tension in the renal cortex (C?PO
2
), outer medulla (M?PO
2
), and renal venous oxygen tension (P
rv
O
2
) were measured by oxygen-dependent quenching of phosphorescence lifetimes of the
systemically infused albumin-targeted (and therefore circulation-confined) phosphorescent
dye Oxyphor G2 13]. Oxyphor G2 has two excitation peaks (?excitation1
?=?440Â nm, ?excitation2
?=?632Â nm) and one emission peak (?emission
?=?800Â 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 13]. For the measurement of renal venous PO
2
(P
rv
O
2
), a mono-wavelength phosphorimeter was used 14]. Oxygen measurements based on phosphorescence lifetime techniques rely on the principle
that phosphorescence can be quenched by energy transfer to oxygen resulting in shortening
of the phosphorescence lifetime. A linear relationship between reciprocal phosphorescence
lifetime and oxygen tension (i.e., the Stern-Volmer relation) allows quantitative
measurement of PO
215].
Renal oxygen delivery and consumption
Arterial oxygen content (AOC) was calculated by (1.31?×?hemoglobin?×?S
a
O
2
)?+?(0.003?×?P
a
O
2
), where S
a
O
2
is the arterial oxygen saturation and P
a
O
2
is the arterial partial pressure of oxygen. Renal venous oxygen content (RVOC) was
calculated as (1.31?×?hemoglobin?×?S
rv
O
2
)?+?(0.003?×?P
rv
O
2
), where S
rv
O
2
is the venous oxygen saturation and P
rv
O
2
is the renal vein partial pressure of oxygen (measured using phosphorimetry). Renal
oxygen delivery was calculated as DO
2
(mL/min)?=?RBF?×?AOC. Renal oxygen consumption was calculated as VO
2
(mL/min)?=?RBF?×?(AOC?–?RVOC).
Renal function
For 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 10Â min. Creatinine clearance rate (CCR) per gram of renal tissue was
calculated with standard formula: CCR [mL/min]?=?(UC
?×?V)/PC
, where UC
is the urine creatinine concentration, V is the urine volume per unit time, and PC
is the plasma creatinine concentration. Renal sodium reabsorption (TNa+
, [mmol/min]) was calculated as TNa+
?=?(PNa+
?×?CCR)???(UNa+
?×?V), where UNa+
is the urine sodium concentration and PNa+
is the plasma sodium concentration.
Renal tissue oxidative stress
Renal tissue malondialdehyde (MDA) levels were determined to assess lipid peroxidation
as a measure of renal oxidative stress. All kidneys were homogenized in cold 5-mM
sodium phosphate buffer. The homogenates were centrifuged at 12,000g for 15 min at 4 °C, and supernatants were used for MDA determination. The level of
lipid peroxides was expressed as micromoles of MDA per milligram of protein (Bradford
assay).
Renal tissue NO levels
NO undergoes a series of reactions in biological tissues leading to the accumulation
of the final products nitrite and nitrate. Thus, the index of the total NO accumulation
is the sum of both nitrite and nitrate levels in the tissue samples. To reduce the
nitrate and nitrate pressnet in the tissue samples to NO, the samples were put in
the reducing agent vanadium (III) chloride (VCl
3
) in 1 mol/L HCl at 90 °C. The VCl
3
reagent converts nitrite, nitrate, and S-nitroso compounds to NO gas which is guided
towards an NO chemiluminescence signal analyzer (Sievers 280i analyzer, GE Analytical
Instruments) allowing the direct detection of NO 16]. Within the reaction vessel, NO reacted with ozone to generate oxygen and excited-state
NO species, of which the decay is associated with the emission of weak near-infrared
chemiluminescence. This signal is detected by a sensitive photodetector and converted
to millivolts (mV). The area under the curve of the detected chemiluminescence (mV?s)
represents the amount of NO-ozone reactions in time and thus the amount of bioavailable
NO in the tested samples. The ratio of tissue NO to tissue protein content was used
for standardization of the NO measurements.
Data analysis
Data analysis and presentation were performed using GraphPad Prism (GraphPad Software,
San Diego, CA, USA). The values are reported as the mean?±?SD. Two-way ANOVA for repeated
measurements with a Bonferroni post hoc test were used for comparative analysis between
groups. A p value of 0.05 was considered statistically significant.