Fig 1.
Virtual grid for random glomerular selection in kidney sections.
A virtual grid was drawn over a kidney section taken at low magnification (40x). This was used to maximize the glomerular selection area and to minimize selection bias. A minimum of two glomeruli were randomly chosen from each sector so that the total number of glomeruli selected for protein analysis is 50 per kidney sample.
Table 1.
Animal characteristics.
Fig 2.
Examples of stained kidney sections used for subjective histological evaluation.
Two randomly chosen slides (4 sections per slide) from each group were evaluated by an independent histopathologist after being stained with hematoxylin and eosin (2A (IH), 2B (IA)) for general glomerular and tubular morphological assessment, cellularity of the glomerulus, and for cellular infiltrates in the cortex and medulla. Glomeruli stained with periodic acid–Schiff stain for glomerular basement membrane and mesangium assessment, and for assessment of glomerular capillary loops and tubular epithelium are shown in Fig 2C (IH) and 2D (IA). Assessments of glomerular and tubular collagen and fibrous tissue were made using Masson's trichrome stain; 2E (IH), 2F (IA). Images taken at 400x with standardized light exposure. IH: Intermittent hypoxia, IA: Intermittent air.
Fig 3.
(3A) The glomerular tuft area was averaged from all available glomeruli per kidney section to give n = 1, using one kidney section per animal. (3B) Mesangial matrix fraction was calculated as the ratio of PAS positive area to total glomerular tuft area and averaged from 50 randomly selected glomeruli per kidney section to give n = 1. (3C) Examples of PAS-stained kidney sections; images taken at 400x magnification. Mesangium indicated as PAS-stained (purple) nuclei-free area within the glomerular tuft area. IA: Intermittent air, IH: Intermittent hypoxia; unpaired t-test, (n = 5).
Fig 4.
Glomerular expression of growth factors by immunohistochemistry.
Semi-quantitative analysis of glomerular expression of TGF-β1 (4A), CTGF (4B) and VEGF-A (4C) proteins by immunohistochemistry. Fig 4D indicates an examples of glomerular protein (CTGF) expression by microscopic viewing. Images taken at 400x magnification; nucleus indicated as blue, while brown staining indicates antigen-antibody reaction inside glomerular cells. Arrows indicate glomerular protein localization. IA: Intermittent air, IH: Intermittent hypoxia; unpaired t-test, (n = 5).
Table 2.
Changes in glomerular proteins expression by immunohistochemistry.
Fig 5.
Proteins semi-quantification in kidney cortex by western blotting.
Semi-quantitative measurements of TGF-β1 (5A), CTGF (5B), VEGF-A (5C), HIF-1α (5D) and Bax/Bcl-2 (5E) proteins in kidney cortex. Protein level was measured from kidney cortex tissue lysate. kDa: Kilodalton, IA: intermittent air, IH: intermittent hypoxia; unpaired t-test, (n = 4–5).
Table 3.
Changes in cortical proteins expression by western blotting.
Fig 6.
Fluorometric in situ cell death detection (TUNEL staining).
Five paraffin fixed kidney sections from each group underwent in situ cell death detection (DNA-strand breaks) using a fluorometric TUNEL staining (red fluorescence) protocol. Slides were counter-stained with DAPI (blue fluorescence) for nuclear detection. The fraction of apoptotic cells was calculated as the number of TUNEL-positive nuclei to the total nuclei per image. Four different images were taken at (200x magnification) from each kidney section to give n = 1. Fig (6A) shows the mean fraction of TUNEL staining-positive cells to the number of available cells. Fig (6B) shows examples of the distribution and amounts of TUNEL positive cells by microscopic viewing. IA: intermittent air, IH: intermittent hypoxia; unpaired t-test, (n = 5).
Fig 7.
Urinary albumin excretion in 24 hours (7A); albumin concentration in urine sample was measured by a standard ELISA method and then normalized to the volume of urine excreted. Serum creatinine was measured by an enzymatic assay and expressed as a concentration in an 8 μl plasma sample (7B). IA: Intermittent air, IH: Intermittent hypoxia; unpaired t-test, (n = 5).