Resveratrol improves renal microcirculation, protects the tubular epithelium, and prolongs survival in a mouse model of sepsis-induced acute kidney injury.

CLP model of sepsis All animals were housed and handled in accordance with the National Institute of Health Guide for the Care of Laboratory Animals with approval from an internal animal care and use committee. CLP was performed on male C57/BL6 mice (Harlan, Indianapolis, IN) aged 39–40 weeks as described previously. 12 , 13 Under isoflurane anesthesia, 1.5 cm of the cecal tip was ligated using a 4-0 silk suture and punctured twice with a 21-gauge needle. An approximately 1-mm column of fecal material was expressed. In sham-operated mice (sham), the cecum was isolated but neither ligated nor punctured. Following surgery all mice received 1 ml of prewarmed saline and were placed in individual cages on a heating pad. Mice studied at time points longer than 6 h were given imipenem/cilastatin (14 mg/kg) and 1.5 ml of normal saline (40 ml/kg, subcutaneous) at 6 h. Administration of RES Fresh solutions of trans -resveratrol (Cayman Chemical Company, Ann Arbor, MI) were prepared in dimethyl sulfoxide (vehicle), kept in the dark, and diluted in normal saline just before use. To assess the very acute effects of RES, mice were administered RES at 5.5 h and studied at 6 h. When mice were studied at 18 h, RES was administered at 6 h post CLP and then again at 12 h because of the relative short half-life of RES in rodents (~30 min 50 , 51 ). For the 48-h survival study, RES was administered through the development of renal injury by dosing at 6, 12, and 18 h. 12 Intravital video microscopy IVVM was performed as described previously. 12 , 13 , 20 Briefly, following anesthesia, FITC-labeled dextran (500 kDa, Sigma, St Louis, MO) and dihydrorhodamine-1,2,3 (DHR, Invitrogen, Carlsbad, CA) were administered through the penile vein to visualize the capillary vascular space and detect RNS generation, respectively. The doses of FITC-dextran and DHR were 1.4 μmol/kg and 0.8 mg/kg, respectively, in 2.1 ml/kg normal saline. The left kidney was exposed by a flank incision and positioned on a glass stage above an inverted Zeiss Axiovert 200M fluorescent microscope equipped with an Axiocam HSm camera (Zeiss, Jena, Germany). Video images of 10 s (approximately 30 frames/s) at ×200 magnification were acquired from five randomly selected, nonoverlapping fields of view. Body temperature was maintained at 36–37 °C with a warming lamp.

Assessment of renal microcirculation

Capillaries were randomly selected from each of the video images collected during IVVM and categorized as ‘continuous flow’ when RBC movement was continuous; ‘intermittent flow’ when RBC movement stopped or reversed; or ‘no flow’ when no RBC movement was observed. Approximately 150 capillaries were analyzed for each animal. Data were expressed as the percentage of vessels in each of the three categories. RBC velocity was calculated in continuously flowing capillaries by measuring the distance traveled by a single RBC over time. Data were expressed as μm/s.

Detection of RNS generation using IVVM

DHR is preferentially oxidized to fluorescent rhodamine by peroxynitrite and perhaps other reactive oxygen species/RNS species but not by superoxide. 30 , 31 Rhodamine fluorescence was visualized at 535 nm excitation and 590 nm emission. Still images exposed for 500 ms were captured from the fields of view used to determine capillary perfusion. Fluorescence intensity was measured by ImageJ (NIH, Bethesda, MD) after first subtracting background fluorescence intensity. Data were expressed as arbitrary units/μm 2 .

Measurement of MAP and HR in conscious mice

MAP and HR were monitored continuously in conscious mice using biotelemetry. Transmitters (Data Sciences International, Minneapolis, MN) were implanted into the carotid artery under isoflurane anesthesia and the animals were allowed to recover for 48 h. Cardiovascular parameters were recorded for 10 s every 5 min. At 5.5 h following surgery, mice were administered RES or vehicle.

Measurement of RBF

Under isoflurane anesthesia, the right renal artery was isolated from the vein and a Transonic Systems (Ithaca, NY)-calibrated 0.5 PSL renal artery Doppler flow probe was positioned around the renal artery. RBF was recorded after the flow stabilized (approximately 10 min after placement of the probe) using PowerLab and LabChart software (AD Instruments, Dunedin, New Zealand). Resveratrol (10 mg/kg) or vehicle was administered through the penile vein. Body temperature was maintained at 36–37 °C with a heating lamp. Data were expressed in ml/min per g kidney weight.

Measurement of GFR

GFR was measured using the single bolus FITC-inulin clearance method. 60 Briefly, a 5% solution was prepared by dissolving FITC-inulin (Sigma, St Louis, MO) in normal saline. The solution was injected through the penile vein at a dose of 3.74 μl/g. Blood (25 μl) was collected into heparinized capillary tubes at 3, 7, 10, 15, 35, 55, 75, 90, and 120 min post injection. FITC-inulin in serum was measured at 485 nm excitation and 538 emission and quantified against a known concentration. Inulin clearance was calculated using a two-phase decay nonlinear regression analysis. GFR was calculated using the fast and slow phases of inulin clearance after normalizing to the combined weight of both kidneys.

Measurement of total serum

NO levels Serum nitrate + nitrite levels were determined using the Total Nitric Oxide Assay Kit (Assay Designs, Ann Arbor, MI) as directed by the manufacturer and concentration expressed as in μmol/l.

Measurement of serum creatinine and blood urea nitrogen concentration

Serum creatinine levels and blood urea nitrogen were measured using the QuantiChrom Creatinine Assay kit and Urea Assay kit, respectively (BioAssay Systems, Hayward, CA).

Immunohistochemistry staining for nitrotyrosine

Nitrotyrosine–protein adducts were detected using a polyclonal anti-nitrotyrosine antibody (Millipore, Billerica, MA) diluted 1:1200 in 1% bovine serum albumin, 0.5% milk in 1 × Tris-buffered saline, pH 7.6, as described previously. 12 Preincubation of the anti-nitrotyrosine antibody with 10 mmol/l nitrotyrosine was used as the nonspecific binding control.

Histology

The periodic acid–Schiff-stained sections were scored in a blinded, semiquantitative manner. For each animal, at least 10 high-power (×400) fields were examined. The percentage of tubules that displayed cellular necrosis, loss of brush border, cast formation, vacuolization, and tubule dilation was scored as follows: 0 = none, 1 = 76%. Survival study Mice subjected to CLP surgery were administered either RES (10 mg/kg, i.p.) or vehicle at 6, 12, and 18 h following CLP and monitored for 48 h. Core body temperature was used as an indicator of pending mortality 35 and was measured every 6 h using a rectal probe. Mice were considered as non-survivors if they died or had to be killed because of two consecutive readings of core temperature below 28 °C.

Statistical analysis

Data, presented as mean±s.e.m., were analyzed using Prism 5.0 (GraphPad Software, San Diego, CA). Before analysis of categorical perfusion, the data were transformed using isometric log ratio transformations. Hotelling’s T 2 -test was then used to calculate test statistics between pairs of groups, and permutation tests were used to calculate P -values for each comparison ( Figure 1a and 4a ). Renal tubular injury scores were analyzed using the nonparametric Kruskal–Wallis test, followed by the Dunn multiple comparison test ( Figure 6d ). Survival curves ( Figure 8 ) were analyzed using a Mantel–Cox log rank test. For all other data, Student’s t -test was used when two groups were compared, and a one-way analysis of variance followed by the Newman–Keuls post-hoc test was used when three or more groups were compared. A P -value