Igure 2k). These final results indicated glomerular damages in indoxyl sulfate-exposed mice.Microarray analysisDifferentiated human podocytes (day 7) were stimulated with 1 mM indoxyl sulfate or 0.1 DMSO for 24 h. After stimulation, total RNA was extracted. Gene expression was analyzed employing a GeneChip Human Gene 1.0 ST Array (Affymetrix, CA, USA). Microarray signals have been normalized using the RMA algorithm. The considerably expressed genes have been chosen depending on ANOVA analysis by Partek Genomics Suite (Partek, St. Charles, MO, USA). The ANOVA gene list was obtained by commercial software program Partek genomic Suite. This Minimum Information regarding a Microarray Experiment-compliant dataset has been deposited inside the NCBI Gene Expression Omnibus, GEO Series accession quantity GSE51834 (http://www.ncbi.nlm.nih.gov/geo/query/ acc.cgitoken=ezanqyoclzunxcf acc=GSE51834).AhR localized to podocyte nuclei in mouse kidneysIndoxyl sulfate is as an endogenous ligand of AhR, acting as a ligand-activated transcription factor that regulates detoxification, carcinogenesis, and inflammation [16,17]. We localized AhR in standard mouse kidneys by using immunofluorescence (Figure 3ac). AhR expression was restricted towards the glomerulus (Figure 3a) within a subset of podocytes good for WT1 and synaptopodin, where it was largely restricted towards the nucleus (Figure 3b and c). Subsequent, we assessed the mRNA expression of Cyp1a1, which is induced by the activation of AhR following ligand binding. Cyp1a1 mRNA expression within the kidney was significantly elevated by two h following exposure and peaked at four h, having a return to typical by 24 h, and was elevated in glomeruli at the same time as entire kidneys at two h following indoxyl sulfate exposure (Figure 3d and e).CTP Epigenetic Reader Domain Statistical analysesThe outcomes had been expressed as imply six SD.Isovitexin In Vivo Data for two groups were analyzed using the Student’s t-test. For many comparisons, evaluation was by ANOVA by utilizing a Bonferroni test (to examine all pairs) or Dunnett test (to evaluate all samples vs.PMID:23891445 the manage samples). Significance was inferred for P,0.05.PLOS One | www.plosone.orgPodocyte Injury by Indoxyl SulfateFigure 1. Indoxyl sulfate induced tubulointerstitial and vascular injuries in mouse kidneys. Serum indoxyl sulfate levels were measured following single dose exposure (a and b). C57BL/6 mice (800 mg/kg, i.p. provided as soon as). n = 1 (time course, panel a). n 7 (240 min, panel b), mean six SD. Histopathology of FVB/N mouse kidneys following chronic exposure to vehicle or indoxyl sulfate, administered at 600 mg/kg/d i.p. for eight w (c ). In contrast to kidneys from vehicle-exposed mice (c), global renal atrophy was observed in 1 of two kidneys from an indoxyl sulfate-exposed mouse (d, arrow). Bars = 500 mm. Vehicle-treated mice manifested histologically unremarkable tubules stained with periodic acid Schiff (e). Inside the macroscopically atrophied kidneys in indoxyl sulfate-exposed mice, prominent tubulointerstitial injury with many, prominent protein casts inside tubules and comprehensive tubular atrophy (f) and foci of interstitial fibrosis have been observed with Masson trichrome staining (g). Bars = 20 mm. doi:ten.1371/journal.pone.0108448.gChronic indoxyl sulfate exposure caused podocyte injury in miceWe characterized podocyte injury in chronically indoxyl sulfateexposed mice following eight w of exposure to indoxyl sulfate (Figure 4a ). Podocytes manifested prominent but incomplete foot approach effacement and podocyte cytoplasmic vacuoles (Figure 4b ) and a focal granular/wrinkled pattern of.
