Supplemental Material (Detailed Methods)

Supplemental Material (Detailed Methods)

Lentivirus productionand intra-renal lentivirus deliveryin vivo

Lentivirus encoding EGFP or EGFP-huA1AR was generated by subcloning EGFP or EGFP-huA1AR (producing a EGFP-A1AR fusion protein, kindly provided by Dr. Raymond B. Penn, Wake Forest University School of Medicine, NC) into a shuttle vector (pLL3.7) and co-transfectingHEK293-FT cells with pLL3.7 expressing EGFP or EGFP-huA1AR, pVSVG (Invitrogen) and p8.9 (from Dr. VanParjs, MIT, Cambridge, MA) utilizing opti-MEM and Lipofectamine2000 as described previously (5,6). In brief, CMV-huA1AR-pLL3.7 and the 2 packaging vectors were cotransfected into 80–90% confluent HEK-293FT cells in 10-cm tissue cultureplates using 20 µl of Lipofectamine 2000 (Invitrogen,Carlsbad, CA) in serum-free OptiMEM medium according to themanufacturer's recommendations. Supernatant was collected after 48 h and passed thorough a 0.45-µm filter to remove cells and debris. An approximate viral titer was determined by infecting HEK293 cells with serial dilutions of the final virus suspension and counting the number of fluorescent cells 48 hr after infection. We typically obtained titers of 2–5 × 106 infectious units/ml starting from one 10-cm plate of HEK293FT cells. In vivo virus transduction to express huA1ARs in the kidneys of mice was performed as described by Nakamura et al. (9) with slight modifications. In anesthetized mice, after temporary occlusion of the left renal pedicle, a 31G needle was inserted at the lower pole of the left kidney parallel to the long axis and was carefully pushed toward the upper pole. As the needle was slowly removed, 100 l filter-purified lentivirus cocktail (EGFP or EGFP-huA1AR, ~5X104 IU/l) was injected. Mice were subjected to hepatic IR 48 hrs after virus injection as described below. Preliminary studies showed that lentiviral-mediated EGFP or EGFP-huA1AR protein as well as mRNA expression in kidney parenchyma were robust after 48 hrs.

Surgery and anesthesia for murine liver IR

After Columbia University Institutional Animal Care and Use Committee approval, male C57 BL/6 mice (25-30 g) were anesthetized with intraperitoneal pentobarbital (50 mg/kg or to effect). Mice were placed under a heating lamp and on a 37°C heating pad. After a midline laparotomy and intraperitoneal application of 500 U of heparin, left lateral and median lobes of the liver were subjected to ischemia with a microaneurysm clip occluding the hepatic triad above the bifurcation. This method of partial hepatic ischemia results in a segmental (~70%) hepatic ischemia but spares the right lobe of the liver and prevents mesenteric venous congestion by allowing portal decompression throughout the right and caudate lobes of the liver(2,4). The liver was then repositioned in the peritoneal cavity in its original location for 60 minutes. The liver was kept moist with gauze soaked in 0.9% normal saline. The body temperature was monitored by an infrared temperature sensor (Linear Laboratories, Fremont, CA) and maintained at 37°C with a heating lamp and a heating pad. After 60 minutes, the liver was reperfused, and the wound was closed.

Vascular permeability of liver and kidney tissues

Changes in liver and kidney vascular permeability were assessed by quantitating extravasation of Evans blue dye (EBD) into the tissue as described by Awad et al. (1) with some modifications. Two percent EBD (Sigma Biosciences, St. Louis, MO) was administeredintravenously at a dose of 20 mg/kg 24 hrs after liver injury. One hr later, mice were killedand perfused through the heart with PBS andEDTA with 10 mL cold saline with heparin (100 U/mL). Liver and kidneys were then removed, allowed to dry overnight at 60 °C, and the dry weights were determined. EBD was extracted in formamide (20 mL/g drytissue; Sigma Biosciences), homogenized, and incubated at 60 °Covernight. Homogenized samples were centrifuged at 12,000 g for30 min and the supernatants were measured at 620 and 740 nm in a spectrophotometer. The extravasated EBD concentration was calculatedagainst a standard curve and the data expressed as micrograms of EBD per gram of dry tissue weight.

F-actin staining of liver and kidney sections

As breakdown of F-actin occurs early after IR, we visualized the F-actin cytoskeleton by staining with phalloidin as an early index of liver as well as renal injury (8). Twenty-four hrs after hepatic IR, liverand kidney tissues were embedded in Tissue-Tek oxytetracycline compound(Fisher Scientific, Pittsburgh, PA) and cut into 5 m sections. To reducebackground staining, the sections were incubated in 1% FBS dissolvedin PBS for 10 min at room temperature.The sections were thenstained with Alexafluor 594 (Red)-labeled phalloidin(Invitrogen, Carlsbad, CA) for 30 min at 37 °C in a humidifiedchamber in the dark. Sections were then washed twice in PBS andmounted with Vectashield (Vector Laboratories, Burlingame, CA). F-actin imageswere visualized with an Olympus IX81 epifluorescence microscope (Tokyo, Japan) and captured and storedusing SlideBook 4.2 software (Intelligent Imaging Innovations Inc., Denver, CO)on a personal computer. The mean fluorescent intensity of F-actin stain from liver or kidney sections after background correction was calculated from 5 random blinded sections with identical surface areas per slide to quantify F-actin degradation after liver IR. We also counted the number of intact liver bile canalicular membranes as well as their fluorescent intensities after F-actin staining (400X fields). To minimize the variations in fluorescent intensity, slides from sham-operated animals and animals subjected to liver were processed together.

Immunohistochemistry for HSP27 and huA1AR

Forty eight hrs after intrarenal lentivirus injection, liver and kidneys were embedded in Tissue-Tek oxytetracycline compoundand cut into 5m sections.The sections were air dried and fixed in 4% paraformaldehyde in PBS and briefly washed in PBS. To reducebackground staining, the sections were incubated in 1% FBS dissolvedin PBS for 10 min. at room temperature. The sections were thenincubated with anti-HSP27 antibody (Abcam, Cambridge, MA) antibody for 1 hr at room temperature. After washing with PBS, the sections were incubated with a goat anti-rabbit Alexa Fluor 594 conjugated secondary antibody (red) for 1hr at room temperature in the dark andmounted with Vectashield (Vector Laboratories, Burlingame, CA). Co-localization (yellow) of EGFP (green – representing either EGFP or EGFP-huA1AR expression) with HSP27 (red) was determined with Z sections taken with a step size of 0.25 m (for total of Z-distance of 5m) with an Olympus Spinning Disk Confocal System microscope and analyzed with the Slidebook software using Peason’s correlation.

To detect the huA1AR expression after EGFP-huA1AR lentivirus injection, we also performed huA1AR immunohistochemistry. Oxytetracycline embedded liver or kidney sections were incubated with anti-huA1AR antibody (1:100 dilution, Affinity BioReagents, Golden, CO) antibody for 1 hr at room temperature. After washing with PBS, the sections were incubated with a goat anti-rabbit Alexa Fluor 594 conjugated secondary antibody (red, 1:100 dilution) for 1 hrat room temperature in the dark andmounted with Vectashield (Vector Laboratories, Burlingame, CA). Co-localization (yellow) of EGFP (green) with huA1AR (red) was determined as described above.

Immunohistochemistry for neutrophils

Paraffin-embedded mouse liver sections were deparaffinized in xylene and rehydrated through a graded ethanol series ending in water. Sections were allowed to sit in 2 changes of phosphate buffered saline (PBS, pH 7.4) for 3 minutes before antigen retrieval. Antigen retrieval was carried out by using pre-heated (95-100 °C) 10 mM Sodium Citrate (pH 6.0, Sigma-Aldrich), and placing the slides above a boiling rack of water for 30 minutes. Endogenous peroxidase activity was quenched with 0.3% H2O2, while non-specific binding was reduced by blocking with 10% normal rabbit serum in PBS containing both Avidin and Biotin (Vector SP-2001).

Avidin was added to the 10% normal rabbit serum to reduce background due to endogenous biotin, biotin-binding proteins or lectins, and incubated with each section for 15 minutes at room temperature. After rinsing each section through 2 changes of PBS for 3 minutes each, Biotin in 10% normal rabbit serum was added to each section and allowed to remain at room temperature for 15 minutes. The Biotin was added to block the remaining Avidin binding sites now present on each tissue section.

Sections were once again rinsed in 2 changes of PBS for 3 minutes each before the primary antibody was added. Slides were then placed in a humidified chamber, and incubated overnight at 4°C with a primary antibody (MCA771G, Serotec, Raleigh, NC) that detects neutrophils, diluted in 2% normal rabbit serum (1:200 dilution) in PBS. Slides were rinsed the next morning in 2 changes of PBS, each lasting 3 minutes, before incubation with the secondary antibody.

Secondary incubation, using horseradish peroxidase–conjugated rabbit anti-rat immunoglobulin G, was done at room temperature for 30 minutes, with the secondary antibody diluted in 2% normal rabbit serum (1:200 dilution, Vector BA-4001) in PBS. Slides were again rinsed in 2 changes of PBS, each lasting 3 minutes, before incubation with ABC reagent.

Incubation with ABC reagent (Vectastain PK-6100) was done for 30 minutes, before the chromogen was developed using freshly made diaminobenzidine (0.5 mg/mL, Sigma-Aldrich) buffered in 0.05 M Tris-HCL (pH 7.4, Sigma-Aldrich) for 2 minutes. A primary antibody that recognized IgG2a (MCA1212; Serotec, Raleigh, NC) was used as a negative isotype control in all experiments, at the same concentration as the primary antibody. Slides were then rinsed in 2 changes of PBS, each lasting 3 minutes, to stop the reaction by removing any last traces of the DAB solution.

The sections were evaluated blindly through the counting of the labeled cells (100X fields).

RNA isolation and RTPCR

Five hours after liver ischemic injury, liver tissue subjected to IR injury was dissected, and total RNA was extracted with Trizol reagent according to the instructions provided by the manufacturer (Invitrogen, Carlsbad, CA). RNA concentrations were determined on the basis of spectrophotometric absorbance at 260 nm, and aliquots were subjected to electrophoresis on agarose gels for verification of equal loading and RNA quality. Semiquantitative RT-PCR was performed to analyze the expression of pro-inflammatory genes (KC, MCP-1, MIP-2, and ICAM-1). The polymerase chain reaction (PCR) cycle number for each primer pair was first optimized to yield linear increases in the densitometric measurements for resulting bands with increasing PCR cycles (15-26 cycles). The starting amount of RNA was also optimized to yield linear increases in the densitometric measurements for resulting bands with the established number of PCR cycles. For each experiment, we also performed semiquantitative RT-PCR under conditions that yielded linear results for glyceraldehyde-3- phosphate dehydrogenase to confirm equal RNA input. On the basis of these preliminary experiments, 0.5 to 1.0 g of total RNA was used as the template for all RT-PCR assays. Primers were designed on the basis of published GenBank sequences for mice. Primer pairs were chosen to yield expected PCR products of 200 to 450 base pairs and to amplify genomic regions spanning 1 or 2 introns to eliminate the confounding effect of amplification of contaminating genomic DNA as described previously. Primers were purchased from Sigma Genosys (The Woodlands, TX). RT-PCR was performed with the Access RT-PCR system (Promega, Madison, WI), which is designed for a single-tube reaction for first-strand complementary DNA synthesis (48°C for 45 minutes) with avian myeloblastosis virus reverse transcriptase and subsequent PCR with Tfl DNA polymerase. PCR cycles included denaturation at 94°C for 30 seconds, annealing at an optimized temperature for 1 minute, and extension at 68°C for 1 minute. All PCR reactions were completed with a 7-minute incubation at 68°C to allow for enzymatic completion of incomplete complementary DNAs. The products were resolved on a 6% polyacrylamide gel and stained with Syber green (Roche, Indianapolis, IN), and the band intensities were quantified with a UVP gel imaging system (Bio-Rad, Hercules, CA).

DNA laddering assay

For DNA laddering, liver and kidney tissues were removed 24 hrs after liver IR, apoptotic DNA fragments were extracted according to the methods of Herrmann et al.(3) and was electrophoresed at 70 V in a 2.0% agarose gelin Tris-acetate-EDTA buffer. This method of DNA extraction selectively isolates apoptotic, fragmented DNA and leaves behind the intact chromatin. The gel was stained with ethidiumbromide and photographed under UV illumination. DNA ladder markers(100 bp) were added to a lane of each gel as a reference for the analysisof internucleosomal DNA fragmentation.

TUNEL staining

For the TUNEL assay, fixed liver and kidney sections obtained at 24 hrs after hepatic IR were deparaffinized in xylene and rehydrated through graded ethanols to water. In situ TUNEL staining was used for detecting DNA fragmentation in apoptosis using a commercially available in situ cell death detection kit (Roche, Nutley, NJ) according to the manufacturer’s instructions.

Caspase 3 immunoblotting

Mouse liver and kidney cortical tissues were obtained at 24 hrs after liver IR or sham operation and were dissected on ice, placed in ice-cold radioimmunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM ethylenediamine tetra-acetic acid, 1% Triton-X, pH=7.4) and homogenized for 10 sec. The samples were then centrifuged for 60 min. at 50,000g, and the resulting supernatant was collected, quantified (for protein concentration), and mixed to a 1x final concentration with Laemmli’s-loading buffer (50mM Tris-HCl, 1% 2-mercaptoethanol, 2% sodium dodecyl sulfate, 0.1% bromophenol blue, 10% glycerol). Equal amounts of protein (50µg) were subjected to electrophoresis through a 15% polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Cleaved caspase 3 expression was subsequently detected by immunoblotting using monoclonal antibody (Santa-Cruz Biotechnologies, Santa-Cruz, CA) diluted 1:500 as described previously (7).

References

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