Imbalance in Renal Vasoactive Enzymes Induced by Mild Hypoxia: Angiotensin-Converting Enzyme Increases While Neutral Endopeptidase Decreases

Figures

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  FIGURE 1. Conventional staining with H-E in renal tissue from normoxic and hypoxic animals. Representative images of kidneys from animals in control (A,B) and hypoxic (C,D) conditions. (A,B) Normoxic tissue has tubules and tubulointerstitial space of normal aspect, both in the medullary zone and in the cortex. Blood vessels present usual appearance. (C,D) In hypoxic kidneys, it is possible to observe tubular dilation in some areas of the field, affecting certain blood vessels that irrigate such areas. Dotted areas correspond to higher magnification images on the right column. G = Glomerulus. Scale bar = 100 μm.

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  FIGURE 2. Conventional staining with PAS in renal tissue from normoxic and hypoxic animals. (A,C) show a general view of cortical and medullary zones for each group. (A,B) correspond to the normoxia group. The color fuchsia is present mainly in the basal membrane and the brush border of proximal tubules. No signs of injury are observed in this group. (C,D) correspond to the hypoxia group, showing an area that presents focal damage. Only a few tubules present dilation, while the area that surrounds it presents normal appearance. Dotted areas indicate higher magnification images presented in the right column. G = Glomerulus. Scale bar = 100 μm.

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  FIGURE 3. Osteopontin in renal tissue from normoxic and hypoxic animals. The arrows indicate the localization of OPN that appears stained in brown. (A,B) correspond to the normoxia group. In (A) it is possible to observe a little OPN stain that indicates low expression levels of this protein. Brown stains are totally absent in (B), and the same happens in others fields of the same renal section, representing normal conditions. (C,D) correspond to the hypoxia group. It is possible to observe an increase of OPN staining, expressed mainly in tubular cells. The left panel shows a cortical area where a glomerulus is observed (G). The right panel corresponds to the outer stripe of the outer medulla. All images have the same magnification. Scale bar = 100 μm.

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  FIGURE 4. Collagen staining with Picrosirius red stain in renal tissue from normoxic and hypoxic animals. (A,B) correspond to the normoxia group. (C,D) correspond to the hypoxia group. The left panel shows a panoramic view of the cortical and medullary zones of both groups. The dotted area indicates images with higher magnification on the right column. The arrows indicate the location of collagen stained in red. Note that peritubular collagen is scarce in all images. However, in the hypoxia group it is possible to observe small areas of fibrosis, characterized by an increase in local collagen. In (A,B) an arterial blood vessel is observed with perivascular collagen, which does not constitute fibrosis. All structures other than collagen are stained in yellow. G corresponds to the glomerulus. Scale bar = 100 μm.

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  FIGURE 5. Kallikrein immunostaining in renal tissue from normoxic and hypoxic animals. Green arrows indicate the localization of kallikrein (brown immunostaining) in both groups. It is expressed only in the cortical zone, specifically in the apical pole and perinuclear area of connecting tubule cells. (A,B) correspond to the normoxia group. Greater expression of kallikrein stains are observed in this group compared to the hypoxia group (C,D), which presents less staining in the same cell type. The immunostained mark is not present in the collecting duct or another tubule type, in the glomerulus (G), or in blood vessels. All images of the cortical zone have the same magnification. Scale bar = 100 μm.

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  FIGURE 6. Kallikrein gene expression in whole kidney tissue samples from normoxic and hypoxic animals. The graph represents the standardized average of kallikrein gene expression in both conditions, expressed as fold change between control versus hypoxic group. Results were averaged and mean values were compiled for statistical analysis. A slight decrease is observed, which does not constitute a statistically significant difference between the normoxia and hypoxia groups. GAPDH was used as a housekeeping gene.

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  FIGURE 7. Angiotensin-converting enzyme (ACE) immunostaining in renal tissue from normoxic and hypoxic animals. ACE localization (immunostained in brown) is indicated with green arrows. It is mainly expressed in the medullary zone, at the brush border of the proximal tubule; more specifically in segment S3. (A,B) correspond to the normoxia group. The ACE stain is less in the control group compared to the hypoxia group (C,D). Additionally, in image (D), the immunostained mark appears in the tubulointerstitial space, where there is also a higher number of infiltrative cells. All images correspond to the medullary zone with the same magnification. Scale bar = 100 μm.

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  FIGURE 8. Angiotensin-converting enzyme gene expression in whole kidney tissue samples from normoxic and hypoxic animals. The graph exhibits the average of ACE gene expression. Units were defined as fold change between control versus hypoxic group. Results were averaged and mean values were compiled for statistical analysis. There is a significant increase (∗∗∗P < 0.001) in ACE gene expression in the hypoxia group when compared to the normoxia group. GAPDH was used as a housekeeping gene.

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  FIGURE 9. Neutral endopeptidase immunostaining in renal tissue from normoxic and hypoxic animals. The NEP protein is indicated with green arrows, and it appears immunostained in brown. Its expression was located in proximal tubules, being more concentrated in the S3 segment (both in superficial and juxtamedullary nephrons) at the medullary ray and outer strip of the medulla, respectively. (A,B) correspond to the normoxia group. NEP expression is higher in the control group compared to the hypoxia group (C,D). All images correspond to the medullary zone, and they have the same magnification. Scale bar = 100 μm.

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  FIGURE 10. Neutral endopeptidase gene expression in whole kidney tissue samples from normoxic and hypoxic animals. The graph shows the average of NEP gene expression. Arbitrary units were defined as fold change between control versus hypoxic group. Results were averaged and mean values were compiled for statistical analysis. There is a significant decrease in NEP gene expression in the hypoxia group compared to the normoxia group (∗∗∗P < 0.001). GAPDH was used as a housekeeping gene.