Figure 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. given once). n = 1 (time course, panel a). n≥7 (240 min, panel b), mean ± SD. Histopathology of FVB/N mouse kidneys following chronic exposure to vehicle or indoxyl sulfate, administered at 600 mg/kg/d i.p. for 8 w (c–g). In contrast to kidneys from vehicle-exposed mice (c), global renal atrophy was observed in 1 of 2 kidneys from an indoxyl sulfate-exposed mouse (d, arrow). Bars = 500 µm. Vehicle-treated mice manifested histologically unremarkable tubules stained with periodic acid Schiff (e). In the macroscopically atrophied kidneys in indoxyl sulfate-exposed mice, prominent tubulointerstitial injury with numerous, prominent protein casts within tubules and extensive tubular atrophy (f) and foci of interstitial fibrosis were observed with Masson trichrome staining (g). Bars = 20 µm.
Figure 2.
Indoxyl sulfate induced glomerular and microvascular injuries in mouse kidneys.
Shown are representative images from FVB/N mice exposed to vehicle (a–c) or indoxyl sulfate (600 mg/kg, i.p. for 8 w), (d–i). 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 changes (d and e) and protein exudate in Bowman's space (d, arrow). In the more severely injured kidneys, glomeruli with increased mesangial matrix/segmental solidification were noted (g, arrow). In the kidneys with more severe injury, occasional glomeruli with mesangiolytic features were 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 µm. The urinary albumin/creatinine ratio (n≥7, mean ± SD) is shown in (j); W denotes weeks after dosing. Podocyte marker mRNA expression in mouse kidneys is expressed as a fold change compared to the vehicle control (k); n≥3, mean ± S.D. * denotes significant differences between the vehicle and indoxyl sulfate groups in the same experiment (P<0.05).
Figure 3.
AhR localized predominantly to podocyte nuclei in mouse kidneys.
Immunostaining of renal cortices from normal C57BL/6 mice for AhR (green) and normal rabbit IgG control (a). The arrows indicate glomeruli containing AhR-positive cells. In the renal cortex, AhR-positive cells were restricted to the glomeruli, and no positive reaction was observed in normal 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 (red), and a merged image with Hoechst nuclear stain (blue) (c). Nuclei expressing AhR and surrounded by synaptopodin-positive cytoplasm, confirming podocyte localization. Bars = 40 µm. The induction of Cyp1a1 mRNA in the kidneys and glomeruli of C57BL/6 mice exposed to vehicle or indoxyl sulfate (800 mg/kg, i.p. given as a single dose). The time course of Cyp1a1 mRNA expression in the kidneys at the time points shown following the final dose as assessed by performing real-time PCR (d). Cyp1a1 mRNA expression in the kidneys and isolated glomeruli at 2 h after dose (e). n≥3, mean ± S.D. * denotes significant differences between the vehicle and indoxyl sulfate groups (P<0.05).
Figure 4.
Indoxyl sulfate treatment induces podocyte injury in mice.
FVB/N mice were exposed to vehicle or indoxyl sulfate (600 mg/kg, i.p.) for 8 w. Transmission electron microscopy images of glomeruli (a–d) demonstrate that indoxyl sulfate exposure is associated with wrinkled and partially collapsed glomerular basement (b and c) and focal podocyte (Pod) foot process effacement (b and c, arrows). Electron-lucent materials were observed in the Bowman space (Bs) in the indoxyl sulfate-exposed mouse (c), and the podocytes contained cytoplasmic vacuoles, consistent with protein resorption droplets (d, arrows). Ery denotes erythrocyte; Cap denotes capillary lumen; Par denotes parietal cell; and Mes denotes mesangial cell. Bars = 1 µm. Representative images of immunostaining of renal cortices are shown (e), bars = 20 µm. Histomorphometry of immune-positive glomerular area fraction that stained for podocin, synaptopodin, vimentin, and AhR in glomeruli are shown (e, f). n≥3, mean ± S.D. * denotes significant differences between the vehicle and indoxyl sulfate groups (P<0.05). A representative immunoblot for podocin, synaptopodin, and β-actin by using whole kidney lysate from kidneys lacking visible atrophy is shown (f); arrowheads indicate the predicted sizes of podocin (42 kDa), synaptopodin (100 kDa), and β-actin (42 kDa). The band intensities were quantified by performing image analysis; n = 7, mean ± S.D (g). * denotes significant differences between the vehicle and indoxyl sulfate groups (P<0.05).
Figure 5.
Indoxyl sulfate activated the aryl-hydrocarbon receptor and altered morphology in mouse podocytes.
RT-PCR analysis for the following genes is shown: Ahr, Aip, and Actb, referring to aryl hydrocarbon receptor, aryl hydrocarbon receptor-interacting protein, and beta actin, respectively (a). M refers to the size marker. 33°C refers to mouse podocytes cultured at 33°C. Day 7 refers to mouse podocytes cultured at 37°C for 7 days. Glo and Kid denote the glomerulus and kidney isolated from an FVB/N mouse. Podocyte marker mRNA expression was measured in mouse podocytes; note that Ahr mRNA expression increased with podocyte differentiation (b). 33°C refers to mouse podocytes cultured at 33°C. Day 7 refers to mouse podocytes cultured at 37°C for 7 days. Day 14 refers to mouse podocytes cultured at 37°C for 14 days. Data generated using real-time PCR; n = 3, mean ± S.D. Fold increase vs. 33°C. * denotes significant differences vs. 33°C (P<0.05). Immunoblotting for AhR in differentiated mouse podocytes demonstrates nuclear translocation following indoxyl sulfate exposure (c). Cyto denotes cytoplasmic protein, Nuc denotes nuclear protein extracted from dimethyl sulfoxide (DMSO)-treated or indoxyl sulfate (IS)-treated mouse podocytes. α-Tubulin and Lamin B were examined to test for protein contamination with cytoplasmic or nuclear proteins, respectively. Each lane contained 20 µg of protein. In a dose-response and time-course study, Cyp1a1 mRNA expression in indoxyl sulfate (IS)-exposed mouse podocytes was measured by real-time PCR (d). n = 3, mean ± S.D. Fold increase vs. each DMSO control. * denotes significant differences vs. DMSO in each time group (P<0.05).Immunofluorescence images of differentiated mouse podocytes exposed to DMSO or indoxyl sulfate for 1 h, 16 h, and 48 h, with staining for AhR (green) and actin (red, phalloidin staining) (e). Indoxyl sulfate exposure is associated with a brief, reversible migration of AhR into the nucleus. Immunoblotting for phosphorylated Rac1/Cdc42 GTPases demonstrated an increase in protein following exposure to indoxyl sulfate for 2 h (f). Each lane contained 5 µg of protein and triplicate wells are shown at 2 h.
Figure 6.
Indoxyl sulfate altered differentiation marker expression in mouse podocytes.
The size of differentiated mouse podocytes decreased with indoxyl sulfate compared to dimethyl sulfoxide (DMSO) control; n = 3, mean ± SD (a). * denotes significant differences between the DMSO and indoxyl sulfate groups (P<0.05). Cell numbers were reduced in indoxyl sulfate-treated mouse podocytes compared to those treated with DMSO; n = 3, mean ± SD (b). Indoxyl sulfate-treated cells were reduced in number at 72 h compared to DMSO control (*, P<0.05). Indoxyl sulfate-treated cells were reduced at 72 h compared to the 8 h (a, P<0.05) and 24 h (b, P<0.05) time points. A dose-response study showed that the viability of differentiated podocytes, assessed using an MTT assay, was reduced to a similar extent at 24, 48, and 72 h, and that the toxic effect reached a plateau at 400 µM; n = 3, mean ± SD (c). The baseline viability was assessed using a 0-µM control for each time group. Podocyte marker mRNA expression was reduced by indoxyl sulfate, as assessed by real-time PCR in differentiated mouse podocytes after indoxyl sulfate treatment (d); n = 3, mean ± S.D. Data are presented as fold increase vs. DMSO (0 µM). * denotes significant differences vs. control for each gene (P<0.05). RNA expression of two cytokines, Il6 and Tnfa, increased in differentiated mouse podocytes after indoxyl sulfate (IS) treatment (e); n = 3, mean ± S.D, fold increase vs. DMSO in each gene. * denotes significant differences vs. DMSO for each time group (P<0.05); h denotes hours after exposure.
Figure 7.
Indoxyl sulfate injures human podocytes.
Immunofluorescence images of autopsied human kidneys shows juxtaposition of AhR in podocyte nuclei surrounded by cytoplasm expressing synaptopodin (a). AhR (red), synaptopodin (green), and normal rabbit IgG control. Immunoblotting for AhR in differentiated human podocytes demonstrates nuclear translocation following indoxyl sulfate exposure (b). Cyto denotes cytoplasmic protein, Nuc denotes nuclear protein extracted from dimethyl sulfoxide (DMSO)- or indoxyl sulfate (IS)-treated human podocytes. Each lane contained 20 µg of protein. Immunofluorescence and phase-contrast images of differentiated human podocytes exposed to DMSO or indoxyl sulfate for 1 h shows rapid nuclear localization following indoxyl sulfate exposure (c). AhR (green). Actin (red, phalloidin staining). Cell viability, assessed using an MTT assay, of indoxyl sulfate-treated differentiated human podocytes was reduced in a dose-dependent and time-dependent fashion compared to DMSO control (d). n = 3, mean ± SD. Baseline viability was assessed using a 0 µM control for each time group. Cell numbers were deceased in indoxyl sulfate-treated differentiated human podocytes compared to DMSO control (e). n = 3, mean ± S.D. Indoxyl sulfate-treated cells were reduced in number at 72 h compared to DMSO control (*, P<0.05). Indoxyl sulfate-treated cell numbers were also reduced at 72 h compared to the 8 h (a, P<0.05) and 24 h (b, P<0.05) time points.
Table 1.
Altered expression of podocyte function-associated genes in indoxyl sulfate-exposed human podocytes.
Table 2.
Altered expression of inflammation-associated genes in indoxyl sulfate-exposed human podocytes.