Supplemental Material (Detailed Methods)
Surgery and anesthesia for murine renal IR, nephrectomy and hepatic 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). Additional pentobarbital was given as needed based
on response to tail pinch. Bilateral flank incisions were made and the left kidney was
subjected to 20 or 30 min. of ischemia with a microaneurysm clip after right
nephrectomy. The duration of ischemia was chosen to produce moderate (20 min.) vs.
severe (30 min.) renal injury in mice. Some mice were subjected to unilateral or bilateral
nephrectomy through bilateral flank incisions.
Liver IR was induced in some mice.
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 (1,2). The liver was then repositioned in the peritoneal cavity in its original
location for 45 min. 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 min., the liver was reperfused, and the wound was closed. During hepatic
ischemia, we performed sham kidney manipulations, 20 min. renal ischemia, unilateral
nephrectomy or bilateral nephrectomy.
Sham operated mice were subjected to
laparotomy and identical kidney or liver manipulations.
Vascular permeability of liver and intestine tissues
Changes in liver and small intestinal vascular permeability were assessed by
quantitating extravasation of Evans blue dye (EBD) into the tissue as described by Awad
et al. (3) with some modifications. Two percent EBD (Sigma Biosciences, St. Louis,
MO) was administered intravenously at a dose of 20 mg/kg after ischemic or nonischemic AKI. 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 small intestine
tissues 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.
Immunohistochemistry for liver and intestine neutrophils, T-lymphocytes and
macrophages
Paraffin-embedded mouse liver and small intestinal (jejunum and ileum) sections
were deparaffinized in xylene and rehydrated through a graded ethanol series ending in
water. Sections used to probe for PMN (7/4) or T-lymphocytes (CD3) were allowed to
incubate in 2 washes of phosphate buffered saline (PBS, pH 7.4) for 3 min. before
antigen retrieval, while sections used to probe for macrophages (F4/80) were allowed to
incubate in 1 wash of phosphate buffered saline with 0.1% Triton-X (PBST, pH 7.4)
instead.
Antigen retrieval for PMN or macrophages was performed in 95°C 10 mM
sodium citrate (pH 6.0, Sigma-Aldrich) for 30 min or 1 min., respectively. Antigen
retrieval for T-lymphocytes was in 95°C 1mM EDTA (pH 8.0, Sigma-Aldrich). Sections
were then placed in a 37 °C incubator for 15 min, before trypsin (1 mg/ml, SigmaAldrich) was added for 10 min. to complete the antigen retrieval process. Endogenous
peroxidase activity for all sections was quenched with 0.3% H2O2, while non-specific
binding was reduced by blocking with 10% normal rabbit serum in PBS or PBST
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 min. at room temperature.
After rinsing each section through 2 washes of PBS or PBST for 3 min. each, biotin in
10% normal rabbit serum was added to each section and allowed to remain at room
temperature for 15 min. The biotin was added to block the remaining avidin binding sites
now present on each tissue section.
Sections were once again washed twice in PBS or PBST for 3 min. 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 (diluted in 2% normal rabbit serum
in PBS or PBST) that either detects neutrophils (PMN, 1:200 dilution, MCA771G,
Serotec, Raleigh, NC), T-lymphocytes (CD3, 1:100 dilution, MCA1477, Serotec,
Raleigh, NC), or macrophages (F4/80, 1:100 dilution, T-2006, Bachem, Torrance, CA).
The next morning, sections were washed twice in PBS before secondary antibody
incubation. Secondary antibody incubation, using horseradish peroxidase–conjugated
rabbit anti-rat immunoglobulin G (1:200 dilution in 2% normal rabbit serum and PBS or
PBST, Vector BA-4001), was performed at room temperature for 30 min. Slides were
again washed twice in PBS or PBST before incubation with ABC reagent.
Incubation with the ABC reagent (Vectastain PK-6100) was performed for 30
min., 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
min. A primary antibody that recognized either IgG2a (for PMN and F4/80, MCA1212,
Serotec, Raleigh, NC) or IgG1 (for CD3, MCA1123, Serotec, Raleigh, NC) was used at
the same concentration as the primary antibody as a negative isotype control for all
experiments. Slides were then washed twice in PBS or PBST to stop the reaction by
removing any last traces of the DAB solution.
The sections were evaluated by blindly counting the labeled cells (100X fields).
RNA isolation and RTPCR
Five hours after ischemic or non-ischemic AKI, liver or small intestinal tissues
were removed 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, TNF-, IL-6, IL-17 and
ICAM-1, Supplemenntal Table). 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 (15-32 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 203 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 min.) 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-min. 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 small intestinal tissues were removed after AKI,
apoptotic DNA fragments were extracted according to the methods of Herrmann et al. (4)
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, formalin fixed liver and small intestinal paraffin sections
obtained after AKI 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.
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