Figure 1.
Renal expression of the endothelin system components in adriamycin nephropathy.
Mice were treated with saline or adriamycin (10 mg/kg) and sacrificed at the indicated time points. RNA was extracted from harvested kidneys and subjected to quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) analysis. The mRNA expression of the ETA receptor (A), ETB receptor (B), and ET-1 (C) is presented. *P < 0.05 compared to 3 week and 5 week adriamycin groups. †P < 0.05 compared to saline controls.
Figure 2.
Atrasentan does not prevent proteinuria in adriamycin nephropathy.
(A) In this prevention protocol, treatment with atrasentan (5 mg/kg i.p., open triangles) was started one day prior to administration of a single dose of adriamycin (ADR, 10 mg/kg i.v., closed triangle) or saline vehicle (Ctrl). Mice were sacrificed 7 days after adriamycin injection, at which time urine, blood, and tissues were collected. (B) Urinary albumin excretion was determined for each mouse and normalized for urinary creatinine excretion (n=11 for Ctrl group, n=12 for ADR group, and n=12 for ADR / ATR group). *P < 0.05 compared to the Ctrl group. (C) Serum creatinine is shown for all mice in a similar fashion. There was no significant difference between groups at this early time point.
Figure 3.
Podocyte injury is not attenuated by atrasentan in the prevention protocol.
Kidneys from the mice treated in Figure 2 were assessed for podocyte markers. (A) Western blot showing no overall change in nephrin levels from whole kidney homogenates. Densitometry was normalized to actin as a loading control. (B) WT1 positive nuclei were counted in 20 glomerular sections for each mouse (n=4) after immunofluorescence staining. *P < 0.05 compared to control. (C) Normal glomerular immunofluorescence staining for nephrin (red) and WT1 (green). Note the linear nephrin staining (white arrow) and abundant WT1-positive nuclei (yellow arrowheads). Scale bar equals 20 μm. (D) Representative micrograph showing the glomerulus from a mouse treated with adriamycin (without atrasentan) and sacrificed at 7 days. Note that the nephrin staining is not uniformly linear and appears speckled in numerous areas (white arrows) and is completely disrupted in other areas (asterisk). There is also a decrease in WT1-positive nuclei (yellow arrowhead). (E) Glomerulus from an adriamycin-treated mouse that has been cotreated with atrasentan as per the protocol in Figure 2A, showing similar losses of linear nephrin staining (white arrow) as well as complete disruption of nephrin (asterisk) and similar reductions in WT-1 nuclei (yellow arrow).
Figure 4.
Atrasentan does not ameliorate established proteinuria.
(A) In this therapeutic protocol, mice were first treated with adriamycin on day 0 (10 mg/kg, closed triangle). After evaluation of albuminuria on day 7, mice were randomized into groups as described in Materials and Methods to receive vehicle (ADR, n=6) or atrasentan at either 7 mg/kg (ADR/ATR 7, n=6) or 20 mg/kg (ADR/ATR 20, n=5) which are designated as open triangles. Atrasentan was started on day 10 and given three times weekly until sacrifice at 35 days. Control mice (Ctrl) that did not receive any adriamycin or atrasentan were also included. (B) Albuminuria at 7 days post-adriamycin. Each dot represents an individual mouse. Note that the mice are grouped such that similar levels of albumin excretion were included in each arm of the study. Albuminuria was then measured at day 21 (C), day 28 (D), and day 35 (E). (F) Serum creatinine was also assessed at 35 days. *P < 0.05 compared to control group only.
Figure 5.
Atrasentan does not ameliorate established podocyte injury in adriamycin nephropathy.
Mice were treated as in Figure 4A. (A, B) qRT-PCR analysis showed nephrin (A) and WT1 (B) mRNA expression. ADR, adriamycin alone; ADR/ATR7, adriamycin-treated mice that also received atrasentan at 7 mg/kg; ADR/ATR20, adriamycin-treated mice that also received atrasentan at 20 mg/kg. (C-F) Western blots and densitometry for nephrin expression in different groups as indicated. (C and E) Atrasentan at 7 mg/kg; (D and F) Atrasentan at 20 mg/kg. (G) WT1-positive nuclei in 20 glomerular sections from each animal (n=3 for each group) were counted as in Figure 3. *P < 0.05 compared to control group only. (H) Representative glomerulus from mouse treated with adriamycin and sacrificed at 35 days (left panel). Atrasentan at a dose of 7 mg/kg (middle panel) or 20 mg/kg (right panel) did not improve the loss of linear staining for nephrin (white arrows), the areas of complete nephrin disruption (asterisk), or the decrease in WT1-positive nuclei (yellow arrowheads). (I) Histologic scoring of 20 glomeruli per animal (n=4 for Ctrl group and n=5 for all other groups) reveals no difference when atrasentan is added to adriamycin treatment. * P < 0.05 compared to control group only. (J) Representative images of glomeruli from Ctrl and treatment groups, as indicated. Note a lack of improvement in glomerular histology with atrasentan treatment. Scale bar equal 50 μm.
Figure 6.
Atrasentan does not affect morphologic injury and fibrogenic gene expression in adriamycin nephropathy.
Masson’s trichrome staining was performed to determine levels of histologic injury in the mice at 35 days after adriamycin injection. A saline-treated control that received neither adriamycin nor atrasentan is shown in Panel A with enlargement of the boxed area in Panel B. Scale bar equals 100 μm in each figure. Histology from a mouse treated with adriamycin is shown in Panel C with corresponding enlargement in D. Adriamycin with atrasentan at 7 mg/kg is shown in Panel E with enlargement in F. Adriamycin with atrasentan at 20 mg/kg is shown in Panel G with enlargement in H. In Panel I, Western blot for α-smooth muscle actin (α-SMA) is shown for different groups as indicated with α-tubulin as a loading control. In Panel J, qRT-PCR was performed for assessing mRNA expression of collagen-I and TGF-β1. * P < 0.05 for ADR / ATR20 compared to saline Ctrl.