Supplemental Material (Detailed Methods)

Lentivirus productionand intra-renal lentivirus delivery in 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-transfecting HEK293-FT cells with pLL3.7 expressing EGFP or EGFP-

huA1AR, pVSVG (Invitrogen) and p 8.9 (from Dr. Van Parjs, MIT, Cambridge, MA)

utilizing opti-MEM and Lipofectamine 2000 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 culture plates using 20 µl of Lipofectamine

2000 (Invitrogen, Carlsbad, CA) in serum-free OptiMEM medium according to the

manufacturer'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

administered intravenously at a dose of 20 mg/kg 24 hrs after liver injury. One hr later,

mice were killed and perfused through the heart with PBS and EDTA 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 dry tissue; Sigma Biosciences), homogenized, and incubated at 60

°C overnight. Homogenized samples were centrifuged at 12,000 g for 30 min and the

supernatants were measured at 620 and 740 nm in a spectrophotometer. The extravasated

EBD concentration was calculated against 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, liver and kidney tissues were embedded in Tissue-

Tek oxytetracycline compound (Fisher Scientific, Pittsburgh, PA) and cut into 5 m

sections.  To reduce background staining, the sections were incubated in 1% FBS

dissolved in PBS for 10 min at room temperature.   The sections were then stained with

Alexafluor 594 (Red)-labeled phalloidin (Invitrogen, Carlsbad, CA) for 30 min at 37 °C

in a humidified chamber in the dark. Sections were then washed twice in PBS

and mounted with Vectashield (Vector Laboratories, Burlingame, CA). F-actin

images were visualized with an Olympus IX81 epifluorescence microscope (Tokyo,

Japan) and captured and stored using 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 compound and cut into 5 m sections.  The

sections were air dried and fixed in 4% paraformaldehyde in PBS and briefly washed in

PBS. To reduce background staining, the sections were incubated in 1% FBS

dissolved in PBS for 10 min. at room temperature.   The sections were then incubated

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 1 hr at room temperature

in the dark and mounted 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 5m) 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 hr at room temperature in the dark

and mounted 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 gel in 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 ethidium bromide and

photographed under UV illumination. DNA ladder markers (100 bp) were added to a lane

of each gel as a reference for the analysis of 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).

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