Intermittent hypoxia causes histological kidney damage and increases growth factor expression in a mouse model of obstructive sleep apnea

Abstract

Epidemiological studies demonstrate an association between obstructive sleep apnea (OSA) and accelerated loss of kidney function. It is unclear whether the decline in function is due to OSA per se or to other confounding factors such as obesity. In addition, the structural kidney abnormalities associated with OSA are unclear. The objective of this study was to determine whether intermittent hypoxia (IH), a key pathological feature of OSA, induces renal histopathological damage using a mouse model. Ten 8-week old wild-type male CB57BL/6 mice were randomly assigned to receive either IH or intermittent air (IA) for 60 days. After euthanasia, one kidney per animal was paraformaldehyde-fixed and then sectioned for histopathological and immunohistochemical analysis. Measurements of glomerular hypertrophy and mesangial matrix expansion were made in periodic acid-Schiff stained kidney sections, while glomerular transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF) and vascular endothelial growth factor-A (VEGF-A) proteins were semi-quantified by immunohistochemistry. The antigen-antibody reaction was detected by 3,3'-diaminobenzidine chromogen where the color intensity semi-quantified glomerular protein expression. To enhance the accuracy of protein semi-quantification, the percentage of only highly-positive staining was used for analysis. Levels of TGF-β, CTGF and VEGF-A proteins in the kidney cortex were further quantified by western blotting. Cellular apoptosis was also investigated by measuring cortical antiapoptotic B-cell lymphoma 2 (Bcl-2) and apoptotic Bcl-2-associated X (Bax) proteins by western blotting. Further investigation of cellular apoptosis was carried out by fluorometric terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) staining. Finally, the levels of serum creatinine and 24-hour urinary albumin were measured as a general index of renal function. Our results indicate that mice exposed to IH have an increased glomerular area (by 1.13 fold, p< 0.001) and expansion of mesangial matrix (by 1.8 fold, p< 0.01). Moreover, the glomerular expressions of TGF-β1, CTGF and VEGF-A proteins were 2.7, 2.2 and 3.8-fold higher in mice exposed to IH (p< 0.05 for all). Furthermore, western blotting protein analysis demonstrates that IH-exposed mice express higher levels of TGF-β1, CTGF and VEGF-A proteins by 1.9, 4.0 and 1.6-fold (p< 0.05 for all) respectively. Renal cellular apoptosis was greater in the IH group as shown by an increased cortical Bax/Bcl-2 protein ratio (p< 0.01) and higher fluorometric TUNEL staining (p< 0.001). Finally, 24-hr urinary albumin levels were higher in mice exposed to IH (43.4 μg vs 9.7 μg, p< 0.01), while there were no differences in serum creatinine levels between the two groups. We conclude that IH causes kidney injury that is accompanied by glomerular hypertrophy, mesangial matrix expansion, increased expression of glomerular growth factors and an increased cellular apoptosis.

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.

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. Histological evaluation.

(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).

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).

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. Renal function index.

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).