Figure 1.
4-month old Long Evans WT rat (Cortex, kidney).
Representative of Long Evans WT kidney. Similarities or differences in KO rats were compared to concurrent age WT controls. A: Hematoxylin and Eosin – no observable abnormalities; B: Lipofuscin (AFIP method) – lipofuscin positive material not observed; C: Chromotrope Aniline Blue – fine granular cytoplasmic staining (red) indicating small amounts of protein in lysosomes of proximal tubular epithelium (open arrow); D: LAMP-1 – diffuse, cytoplasmic, fine granular staining in proximal tubular epithelium; E: LAMP-2 – intense staining of apical cytoplasm of proximal tubular epithelium; F: NAGLU – fine granular cytoplasmic staining of proximal tubular epithelium. Scale bar = 0.5 mm
Figure 2.
1-month old LRRK2 KO rat (Cortex, kidney).
A: Hematoxylin and Eosin – no observable abnormalities; B: Lipofuscin (AFIP method) – lipofuscin positive material not observed; C: Chromotrope Aniline Blue – CAB positive staining not readily visible and similar to WT rat; D: LAMP-1 – diffuse, cytoplasmic, fine granular staining with a slight increase in intensity in proximal tubular epithelium; E: LAMP-2 - intense staining of apical cytoplasm in proximal tubular epithelium similar to WT control; F: NAGLU – fine, diffuse, granular cytoplasmic staining and apical staining in proximal tubular epithelium similar to WT control (the 1, 2 and 8 month old WT rats in the second cohort had similar baseline NAGLU staining that was slightly different (apical and diffuse cytoplasmic) from those rats in the first cohort (diffuse cytoplasmic). Scale bar = 0.5 mm
Figure 3.
2-month old LRRK2 KO rat (Cortex, kidney).
A: Hematoxylin and Eosin – slight increase in size and variability of hyaline droplets in proximal tubular epithelium (open arrow); B: Lipofuscin (AFIP method) – Lipofuscin positive material not observed; C: Chromotrope Aniline Blue – increased size, number and variability of hyaline droplets in proximal tubular epithelium (open arrows). Hyaline droplet variability was more easily identified using CAB staining when compared to H&E stains; D: LAMP-1 – increased cytoplasmic staining intensity in proximal tubular epithelium; E: LAMP-2 – increased globular cytoplasmic staining (solid arrows) with apical cytoplasmic staining- persisting in proximal tubular epithelium; F: NAGLU – increased globular staining in proximal tubular epithelium (solid arrow). Scale bar = 0.5 mm
Figure 4.
4-month old LRRK2 KO rat (Cortex, kidney).
A: Hematoxylin and Eosin – brown, globular pigment (dotted arrows) and intracytoplasmic, clear vacuoles in proximal tubular epithelium with occasional hyaline droplets (open arrow); B: Lipofuscin (AFIP method) – positive dark red staining of pigment (dotted arrows) and clear vacuoles in proximal tubular epithelium; C: Chromotrope Aniline Blue – Increase in number, size and variability of hyaline droplets (red, open arrows) and clear cytoplasmic vacuoles in proximal tubular epithelium; D: LAMP-1 – Globular cytoplasmic staining in proximal tubular epithelium (solid arrows); E: LAMP-2 – increased globular cytoplasmic staining (solid arrows) with apical staining persisting in proximal tubular epithelium; F: NAGLU – increase intensity of globular cytoplasmic staining (open arrows). Scale bar = 0.5 mm
Figure 5.
8-month old LRRK2 KO rat (Cortex, kidney).
A: Hematoxylin and Eosin – increased amounts of brown pigment (dotted arrows) with cytoplasmic vacuolation in proximal tubular epithelium; B: Lipofuscin (AFIP method) – positive dark red staining of pigment in proximal tubular epithelium (dotted arrows); C: Chromotrope Aniline Blue – hyaline droplets (red open arrows) are smaller and not as variably-sized. Hyaline droplets are occasionally noted along with brown pigment in proximal tubular epithelium; D: LAMP-1 – Increased globular cytoplasmic staining (solid arrows) in proximal tubular epithelium; E: LAMP-2 – increased globular cytoplasmic staining with increased intensity (solid arrow) and persistence of apical cytoplasmic staining; F: NAGLU – globular cytoplasmic staining is increased in intensity in proximal tubular epithelium (solid arrows). Scale bar = 0.5 mm
Figure 6.
12-month old LRRK2 KO rat (Cortex, kidney).
A: Hematoxylin and Eosin – increased amounts of brown pigment (dotted arrows) in proximal tubular epithelium; B: Lipofuscin (AFIP method) – positive dark red staining of pigment (dotted arrows); C: Chromotrope Aniline Blue – positive red hyaline droplets are small and not as variable (open arrows) and are observed in proximal tubular mostly separate from brown pigment (dotted arrows); D: LAMP-1 – increased globular cytoplasmic staining (solid arrows); E: LAMP-2 – increased globular cytoplasmic staining with increased intensity (solid arrows) and persistence of apical cytoplasmic staining; F: NAGLU – increase in intensity of globular cytoplasmic staining (solid arrows). Scale bar = 0.5 mm
Figure 7.
8-month old LRRK2 KO rat (Cortex, kidney).
WT: Kidney Injury Molecule-1 (KIM-1) staining in the Long Evans WT rat. There is focal positive staining (solid arrows) within tubular epithelial cytoplasm in an area of chronic progressive nephropathy; KO: KIM-1 globular staining is observed in proximal tubular epithelium (solid arrows) which colocalized with brown pigment noted on H&E sections. Scale bar = 0.5 mm
Figure 8.
Morphological changes in the kidney of 16-month old LRRK2 KO rats vs.
WT.
(A) Increased number of lysosomes and electron dense material (black arrows) in KOs compared to WT (white arrow) (B) Accumulation of lipid droplets in glomerulus of KO animals compared to WT, which have no lipid droplets. N: Nucleus. Scale bar = 2 µm
Figure 9.
Accumulation of Lamellar Bodies in Type II Alveolar Cells in the lung of 16-month old LRRK2 KO rats vs. WT.
(A) Graphs depicting Number of Lamellar Bodies in Type II Alveolar Cells and Area of Type II Alveolar Cells. (WT n=4, KO N=4) * - denote significance by Students t-test, P<0.05. Values are means + Standard Error of the Mean. (B) Normal Lamellar Body morphology (white arrow) in WT compared to proliferation of lamellar bodies (black arrow) in KO. N: Nucleus. Scale bar = 2 µm
Figure 10.
Accumulation of lipid droplets in hepatic cells in 16-month old LRRK2 KO rats vs. WT.
(A) Graphs showing changes in number of lipid droplets and density of lipid droplets per hepatocyte. (WT n=2, KO n=4) *- denote significance by Student’s t-test, P<0.05. Values are means + Standard Error of the Mean. (B) Comparison of normal accumulation of lipid droplets (white arrow) in hepatic cells in WT to increased accumulation in hepatic cells (black arrow) in KOs. N: Nucleus. Scale bar = 2 µm