Nd synaptopodin protein levels weresignificantly reduced in the indoxyl sulfate-treated mice not displaying macroscopic renal atrophy (Figure 4g).Indoxyl sulfate activated AhR and perturbed the actin cytoskeleton in cultured mouse podocytesmRNA expression of AhR and its partner AhR-interacting protein 2 (Aip2) was detected in mouse glomeruli and kidneys also as cultured mouse podocytes, as well as the AhR mRNA expression level considerably enhanced with podocyte differentiation (Figure 5a and b). Following exposure to 1 mM indoxyl sulfate, nuclear translocation of AhR was clearly observed at 30 min by utilizing proteins extracted from nuclei as well as the cytoplasm, and nuclear AhR was nonetheless observed at 60 min by immunoblotting (Figure 5c). Cyp1a1 mRNA expression was induced beginning two h immediately after indoxyl sulfate exposure, and this boost was sustained forPLOS One | plosone.orgPodocyte Injury by Indoxyl SulfateFigure two. Indoxyl sulfate induced glomerular and microvascular injuries in mouse kidneys. Shown are representative photos from FVB/N mice exposed to car (a ) or indoxyl sulfate (600 mg/kg, i.p. for eight w), (d ). In comparison to histologically unremarkable glomeruli (a), arterioles (a, arrow, and b) and arteries (c) in vehicle-exposed mice, glomeruli in indoxyl sulfate-exposed mice showed ischemic alterations (d and e) and protein exudate in Bowman’s space (d, arrow). Inside the additional severely injured kidneys, glomeruli with improved mesangial matrix/segmental solidification were noted (g, arrow). Within the kidneys with much more serious injury, occasional glomeruli with mesangiolytic options have been present (h and i). Histologically unremarkable mid-sized artery in vehicle-exposed mice, reduplication of elastic lamina mid-sized artery in indoxyl sulfate-exposed mouse are shown (f). Occasional glomerular arterioles demonstrated arteriosclerosis (i, arrow). Bars = 20 mm. The urinary albumin/creatinine ratio (n 7, imply 6 SD) is shown in (j); W denotes weeks soon after dosing. Podocyte marker mRNA expression in mouse kidneys is expressed as a fold modify in comparison with the automobile handle (k); n 3, imply 6 S.D. * denotes important differences between the vehicle and indoxyl sulfate groups in the same experiment (P, 0.05). doi:10.1371/journal.pone.0108448.g24 h with 0.1 mM indoxyl sulfate and for 72 h with 1.0 mM indoxyl sulfate (Figure 5d). Utilizing immunofluorescence staining (Figure 5e), we observed scant AhR protein in the nucleus or in perinuclear places of DMSO handle podocytes. Following 1.0 mM indoxyl sulfate exposure,distinct and intense AhR staining was observed inside the nucleus at 60 min and was no longer present at 16 h in spite of continued exposure.2-Bromo-4-formylnicotinonitrile site Indoxyl sulfate exposure caused morphological changes within the mouse podocytes, inducing a additional fusiform shape and reorganization from the actin cytoskeleton from stress fibers toPLOS A single | plosone.179056-94-1 custom synthesis orgPodocyte Injury by Indoxyl SulfateFigure 3.PMID:23509865 AhR localized predominantly to podocyte nuclei in mouse kidneys. Immunostaining of renal cortices from standard C57BL/6 mice for AhR (green) and regular rabbit IgG handle (a). The arrows indicate glomeruli containing AhR-positive cells. In the renal cortex, AhR-positive cells have been restricted towards the glomeruli, and no good reaction was observed in regular rabbit IgG controls. Immunostaining for WT1 (green), AhR (red), and a merged image with Hoechst nuclear stain (blue); some nuclei are yellow, suggesting that podocyte nuclei contain AhR (b). Immunostaining for synaptopodin (green), AhR (.