Acute kidney injury (AKI) is frequently complicated by extra-renal multi-organ injury including intestinal and hepatic dysfunction. initiate intestinal and hepatic injury by hepatic and systemic delivery of IL-17A by macrophages. Modulation of Paneth cell dysregulation may have therapeutic implications by reducing systemic complications arising from AKI. method. Table 1 Primers used to amplify mRNAs encoding mouse GAPDH, IL-17A and cryptdin 1 based on published GenBank sequences for mice. Respective anticipated RT-PCR product size, PCR cycle number for linear amplification and annealing temperatures used for each primer … Vascular permeability of liver and intestine tissues 102841-43-0 supplier Changes in liver, kidney and small intestine vascular permeability were assessed by quantitating extravasation of Evans blue dye (EBD) into the tissue as described by Awad TUNEL staining was used for detecting DNA fragmentation in apoptosis using a commercially available cell death detection kit (Roche, Nutley, NJ) according to the manufacturers instructions. We further confirmed that the TUNEL positive cells are endothelial cells by staining serial small intestine (jejunum) sections with 102841-43-0 supplier TUNEL and CD34 (an endothelial cell marker, Abcam Inc., Cambridge, MA). For DNA laddering, apoptotic DNA fragments were extracted according to the methods of Herrmann (29) 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. Laser capture micro-dissection (LCM) of Paneth cells LCM of individual Paneth cells was performed with the PixCell I LCM System (Arcturus Executive, Mountain View, Calif., USA) as described (30). Small intestines were embedded in Optimum Cutting Heat 102841-43-0 supplier (OCT) compound (Sakura, Torrance, CA), sectioned at a thickness of 10 M and mounted TSPAN7 on 1.0 Pencil Membrane Slides (Carl Zeiss, Thornwood, NY). The sections were then prepared for micro-dissection using an LCM staining kit (Ambion, Austin, Tx) through a graded alcohol series (95%, 75%, 50%) followed by cresyl violet staining. After de-staining via second graded alcohol series (50%, 75%, 95%), they were dehydrated in 100% ethanol followed by xylene. LCM was performed on a Zeiss Axiovert 200M microscope equipped with PALM RoboSoftware (Carl Zeiss, Thornwood, NY) and the total area of tissue collected per slide was tracked and recorded. RNA was isolated from the dissected tissue by following the protocol 102841-43-0 supplier provided by the RNAqueous-Micro kit (Ambion, Austin, TX) via column purification. Electron Microscopy Small intestines were fixed in 4% paraformaldehyde/3% glutaraldehyde in 10 mM sodium phosphate buffer (pH 7.4) for 48 hrs. All samples were postfixed with 1% osmium tetroxide in 100 mM cacodylate buffer (pH 7.4) on ice for 1 hr. Samples were then treated with 0.5% aqueous uranyl acetate, dehydrated in graded alcohol, treated with propylene oxide, and embedded in Embed 812 (Electron Microscopy Sciences). The resin was polymerized in a 60C oven for 2-3 days. Sections were cut with a Dupont diamond knife in Reichert-Jung UltraCut At the ultramicrotome, collected on copper mineral grids, and doubly stained with saturated aqueous uranyl acetate and lead citrate. Ultrathin sections were imaged for Paneth cells using a JEM-1200EX electron microscope manufactured by JEOL. Isolation of intestinal crypts Intact small intestinal crypts were isolated with the distended intestinal sac method as described by Traber (31) with slight modifications. Small intestine from the duodenum to the ileum was removed and rinsed thoroughly with intestinal wash answer [0.15 M NaCl, 1 mM dithiothreitol (DTT), and 40 pg/mL phenylmethylsulfonyl fluoride (PMSF)] and then filled with buffer A [(in mM) 96 NaCl, 27 sodium citrate, 1.5 KCl, 8 KH2PO4, 5.6 Na2HPO4, and 40 pg/mL PMSF (pH 7.4)]. The ends were clamped with micro-clips and the intestine was filled to a pressure of 50 cmH2O. The filled intestine was submerged in oxygenated 0.15 M NaCl at 37C for 40 min., drained and the answer was discarded. The intestine was then packed with buffer W [(in mM) 109 NaCl, 2.4 KCl, 1.5 KH2PO4, 4.3 Na2HPO4, 1.5 EDTA, 10 glucose, 5 glutamine, 0.5 DTT, and 40 pg/mL PMSF (pH 7.4)], incubated at.