Supplementary MaterialsFigure S1: qPCR validation of a subset of differentially expressed
Supplementary MaterialsFigure S1: qPCR validation of a subset of differentially expressed genes in all mutant versus control kidneys. Hdac target genes. Fold change of mRNA expression of 5 genes in (mutant) kidneys versus controls. *p?=? 0.05, ***p?=?0.0005.(TIF) pone.0072762.s005.tif (288K) GUID:?9F3EEB41-EB9A-4799-A5D0-6CEE5D40D67E Figure S6: qPCR validation of a subset of differentially expressed sexually dimorphic genes. Fold change of mRNA expression of 14 genes in (mutant) kidneys versus controls. *p?=? 0.05, **p?=? 0.005, ***p?=?0.0005.(TIF) pone.0072762.s006.tif (501K) GUID:?204BA7C9-4BFB-4617-B6A6-934991EABFDE Figure S7: qPCR validation of a subset of urothelium specific genes between mild and severe kidneys. Fold change of mRNA expression of 11 genes in severe kidneys versus mild kidneys. *p?=? 0.05, **p?=? 0.005, ***p?=?0.0005.(TIF) pone.0072762.s009.tif (472K) GUID:?4F1B2FBE-9620-45D8-AE9E-29D2FBA63145 Table S1: Genes with 2-fold differential expression between all mgb?/? and wildtype kidneys. (XLS) pone.0072762.s010.xls (90K) GUID:?D5085F5E-A647-44D7-8B4B-A111F4AF2D14 Table S2: Top Toxicological Functions Identified by IPA of All Mutant Kidneys to Controls. (DOC) pone.0072762.s011.doc (30K) GUID:?25ECA9E4-02F2-438A-B704-CFD51ED6570C Table S3: Differentially Expressed TGF-/Smad Target Genes Comparing All Mutant and Control Kidneys. (XLS) pone.0072762.s012.xls (22K) GUID:?090D6B49-C438-4418-B17C-1CF434A34858 Table S4: Differential Gene Expression Between Mild and Severe Hydronephrotic mgb?/? Kidneys. (XLS) pone.0072762.s013.xls (2.0M) GUID:?355C0C33-38AC-4D4F-86C6-2FB70EB06CFA Abstract Congenital obstructive nephropathy is a common cause of chronic kidney disease and a leading indication for renal transplant in children. The cellular and molecular responses of the kidney to congenital obstruction are incompletely characterized. In this study, we evaluated global transcription in kidneys with graded hydronephrosis in the (versus kidneys identified the expression of several novel candidate markers of renal injury. This study indicates how the advancement of intensifying hydronephrosis in mice advances at variable rates, such that by 3C4 weeks of age, a spectrum of obstruction occurs associated with mild to severe reductions in renal parenchyma and function . This variability parallels that seen in humans with obstructive uropathy . In this study, we utilized this variability to evaluate the global transcriptomes of male mice were subject to renal ultrasound to establish the degree of hydronephrosis as previously published . Of note, our method of grading hydronephrosis is based on the degree of parenchymal preservation, and does not directly correlate CX-5461 manufacturer with clinical grading of hydronephrosis, such as the Society of Fetal Urology Grading System (http://www.sfu-urology.org/sfu_hydrone_grading.cfm). Kidneys with 67% parenchyma were considered mildly, 34C66% moderately and 33% severely affected. Mice were euthanized, kidneys extracted, snap frozen and stored at ?80C. RNA Extraction and Microarray Hybridization Total RNA was extracted using mirVana? kit (Life Technologies, Carlsbad, CA). RNA integrity was analyzed using Agilent 2100 Bioanalyzer Lab-On-A-Chip 6000 Series II chip (Agilent Technologies, Santa Clara, CA). Samples were hybridized to Agilent SurePrint G3 Mouse GE 860 K Microarray and scanned using CX-5461 manufacturer Agilent G2505C Microarray Scanner. Raw data were quality-assessed, filtered for outliers and normalized to remove nonbiological variation. Differential gene expression was defined Rabbit Polyclonal to BMP8B using a 10% false discovery rate (FDR) and adjusted as an endogenous control. Results were expressed using the 2 2?CT method by normalizing to a common pool of control kidney cDNA . The average fold change standard error was graphed, and test. Immunohistochemistry (IHC) Formalin fixed, paraffin embedded kidneys were sectioned at 4 m. Deparaffinized sections were rehydrated, subjected to antigen retrieval, peroxidase block, biotin block, and CX-5461 manufacturer Superblock (Scytek, Logan, UT). Primary antibodies were incubated for 1 hour at the following dilutions: anti-Upk3a (Research Diagnostics Inc., Flanders, NJ.) 1500; anti-Krt14 (Covance, Princeton, NJ) 11600; anti-Ki-67 (Abcam, Cambridge, MA) 1800. Biotinylated secondary antibody and HRP-conjugated streptavidin were implemented (Scytek). Slides were developed using diaminobenzamide (MP Biomedicals, Santa Ana, CA), counterstained with hematoxylin and visualized using an Olympus BX-51 microscope (Olympus, America, Center Valley, PA). Ki-67 Quantification Ki-67 positive and Ki-67 negative nuclei within the renal urothelium were identified using anti-Ki-67 labeled and hematoxylin counterstained CX-5461 manufacturer tissue CX-5461 manufacturer sections. Longitudinally oriented, four-micron thick kidney sections were collected near the hilum. Briefly, contours were traced onto morphometric tissues of interest; kidney.