Supplemental Material (Detailed Methods) s1

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 non-ischemic 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, Sigma-Aldrich) 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-a, 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 mg 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.

References

1. Chen SW, Park SW, Kim M, Brown KM, D'Agati VD, and Lee HT (2009). Human heat shock protein 27 overexpressing mice are protected against hepatic ischemia and reperfusion injury. Transplantation 87:1478-1487.

2. Kim J, Kim M, Song JH, and Lee HT (2008). Endogenous A1 adenosine receptors protect against hepatic ischemia reperfusion injury in mice. Liver Transpl 14:845-854.

3. Awad AS, Ye H, Huang L, Li L, Foss FW, Jr., Macdonald TL, Lynch KR, and Okusa MD (2006). Selective sphingosine 1-phosphate 1 receptor activation reduces ischemia-reperfusion injury in mouse kidney. Am J Physiol Renal Physiol 290:F1516-F1524.

4. Herrmann M, Lorenz HM, Voll R, Grunke M, Woith W, and Kalden JR (1994). A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res 22:5506-5507.