Table 1.
Primary antibodies used in the study.
Figure 1.
Aldosterone induces accumulation of punctate RPL22 immunoreactivity in distal renal tubules.
A) Negative immunostaining of renal cortex from control rats (H-95, Table 1). B) Punctate immunolabeling of renal cortex from aldosterone treated rats with the same antibody. C) Sections from aldosterone plus spironolactone treated rats were stained in parallel to those shown in A and B. Kidney sections from aldosterone treated rats were double stained with H-95 and RPL22 antibodies. D) Coomassie blue stained gel showing the eluates from immuno-precipitation experiments with the H-95 antibody. Ctrl are controls, Aldo are aldosterone treated rat samples. E) Single channel signal from RPL22 staining. F) Single channel signal from H-95 staining. G) The merged channels (H-95 red, RPL22 green) overlaid on differential interference contrast image (DIC). “PT” marks proximal tubules, while “DT” indicates distal renal tubules and connecting tubules.
Table 2.
LC MS/MS analysis of proteins pulled down by H95 preferentially in samples from aldosterone treated rats but not detected in IP without beads or without antibody.
Figure 2.
Identification of the tubular segment and cell structures displaying aldosterone-induced punctate immunoreactivity.
Example of double immunofluorescence staining with RPL22 (green) and antibodies against tubule markers (all in red): NKCC2 (A), NCC (B), calbindin-D28K (C), H+-ATPase (D), and AQP2 (E & F) in kidney cortex from aldosterone treated rats. Fluorescence signals are overlaid on the corresponding DIC image.
Figure 3.
Immuno-gold electron micrographs of anti-RPL22 stained cryo-sections.
Top left panel is a cellular overview of a distal renal tubule and the red box indicates the area magnified in left bottom panel. Here, the red box corresponds to the highest magnification electron micrograph (right panel). Arrows point to gold particles.
Figure 4.
RPL22 colocalizes with aggregates containing the proteasome 20 s subunit.
Double labeling immunofluorescence staining was performed on sections of paraffin embedded tissue from aldosterone treated animals for RPL22 and organelle markers from the endocytotic pathway or proteasome subunit. A) Co-labeling of the accumulated protein (red) and an early endosome marker, anti-EEA1 (green) overlaid on DIC image. B) Co-labeling with a recycling endosome marker, anti-Rab11 (green) with DIC overlay. C) Co-labeling with a lysosome marker, anti-cathepsin D (green). D) The immunostaining pattern of the marker anti-proteosome 20 s (green). E) Overlay with RPL22 fluorescence signals (red) and the corresponding DIC image. Yellow color indicates colocalization. F) Quantitation of co-localization of the accumulated immunoreactive protein with the vesicular markers (mean values from four images from five aldosterone treated animals).
Figure 5.
RPL22 colocalizes with the proteasome 20 s subunit by electron microscopy.
Immuno-gold electron microscopy identifies the subcellular co-localization of RPL22 and proteasome subunit 20 s. Double labeling immuno-gold electron micrographs applying the H-95 antibody against aggregated RPL22 protein and proteasome 20 s on cryo-sectons. Top left panel is a cellular overview of a distal renal tubule and the red box indicates the area magnified in left bottom panel. The red box corresponds to the highest magnification electron micrograph bottom panel). Arrows point to 10 nm gold particles (proteasome 20 s) and arrowheads point to 5 nm gold particles (RPL22).
Figure 6.
Aldosterone administration increases proteasome numbers and labeling intensity in distal renal tubules.
A) Double labeling for proteasomes (proteasome 20 s, green) and a marker for DCT (NCC, red) in renal cortex from control rats. B) Similar fluorescence labeling in renal cortex from aldosterone treated rats. C) Quantitation of the mean number of proteasome-containing punctae, the mean area of these, and the mean proteasome 20 s immunoreactivity in the control and aldosterone treated groups in DCT (Con and Aldo, as indicated, n = 5). D) Double labeling of proteasomes (proteasome 20 s, red) and a marker for CNT (calbindin-D28K, blue) in renal cortex from control rats. E) Similar fluorescence labeling in renal cortex from aldosterone treated rats. F) Quantitation of the mean number of proteasome-containing punctae, the mean area of these, and the mean proteasome 20 s immunoreactivity in the control and aldosterone treated groups in CNT (Con and Aldo, as indicated, n = 5). * indicates statistical significance.
Figure 7.
Aldosterone administration does not change HDAC6 staining in renal tubules.
A) Double labeling for histone deacetylase 6 (HDAC6, green) and a marker for DCT (NCC, red) in renal cortex from control rats. Presence of brush border was used as selection criterion for PT. B) Similar fluorescence labeling in renal cortex from aldosterone treated rats. C) Quantitation of the mean number of HDAC6 punctae, the mean area of these, and the mean HDAC6 immunoreactivity in the control and aldosterone treated groups in PT (Con and Aldo, as indicated, n = 5). D) Comparizon of the mean number of HDAC6 punctae and the mean area of these in DCT and PT. E) Quantitation of the mean number of HDAC6 punctae, the mean area of these, and the mean HDAC6 immunoreactivity in the control and aldosterone treated groups in DCT (Con and Aldo, as indicated, n = 5).F) Double labeling histone deacetylase 6 (HDAC6, green) and a marker for CNT (calbindin-D28K, red) in renal cortex from control rats. G) Similar fluorescence labeling in renal cortex from aldosterone treated rats. H) Quantitation of the mean number of HDAC6 punctae, the mean area of these, and the mean HDAC6 immunoreactivity in the control and aldosterone treated groups in CNT (Con and Aldo, as indicated, n = 5). * indicates statistical significance.
Figure 8.
Analysis of the colocalization between proteasome 20 s aggregates and HDAC6.
A) Triple fluorescence labeling for proteasome 20 s (red), aggresome (HDAC6, green), and calbindin-D28K (blue) merged with the corresponding DIC image. B) Magnification of tubular structures in the same image. C) Quantitation of the co-localization of proteasome 20 s immunoreactive punctae and the aggresome transfer protein HDAC6 (n = 5, n.s.).
Figure 9.
Co-localization of proteasome 20 s aggregates and HDAC6 after aldosterone administration.
A) Double fluorescence labeling for RPL22 aggregates (H-95, red) and HDAC6 (green) in kidney from control rat. B) Similar labeling in kidney from aldosterone treated rat. Arrow heads indicate colocalization (in yellow). C) The immunostaing for the de-ubiquitinase ataxin-3 in control rat kidney sections. D) Representative micrograph of renal cortex of an aldosterone treated rat stained for ataxin-3. E) Double fluorescence labeling for aggresome initiator protein p62 (red) and HDAC6 (green) in kidney from control rat. F) Similar labeling in kidney from aldosterone treated rat. G) Renal cortex from control rats was labeled for autophagosomes by anti-LC3 antibodies (green) and overlaid on DIC image. H) Similar fluorescence labeling of renal cortex from aldosterone treated rat. I) Similar staining where distal renal tubules were identified by calbindin-D28K (red). J) Corresponding micrograph from aldosterone treated rat. “PT” marks proximal tubules while “DT” are distal renal tubules and connecting tubules.
Figure 10.
Distal renal tubular vimentin expression and cell infiltration.
A) Typical vimentin immunoreactivity in control rat kidney. B) Similar staining of renal cortex from aldosterone treated rats. C) Lymphocyte infiltration was sometimes observed in the same areas where tubular vimentin expression was detected (dotted outline). D) Double fluorescence labeling for vimentin (red) and the cell-cell adhesion protein E-cadherin (green). E. Vimentin staining of a rat kidney cortex from aldosterone treated rat. F) Similar staining of a rat co-administered with aldosterone and spironolavctone from the same experiment. “Glom” are glomeruli, “BV” show blood vessels, “PT” marks proximal tubules, while “DT” indicates distal renal tubules and connecting tubules.
Figure 11.
Model of the cellular protein degradation systems.
After aldosterone treatment, distal renal tubular cells appear to have inadequate proteasomal capacity to degrade ubiquitinated protein (1) to short peptides (2). Instead, some cytosolic proteins, such as RPL22 (3) form aggregates with the proteasome subunits. In the absence of ataxin-3 (4), protein aggregates are not transferred by HDAC6 (5) to the aggresome (7) and aggregates can therefore not be cleared by autophagy.