The tissues were fixed in a solution of 10% formaldehyde for at least 24 h. Renal tissue was embedded in paraffin. Tissue sections (5 µm) were prepared using a microtome and mounted on slides. Masson’s trichrome stain (Merck KGaA, Darmstadt, Hesse, Germany) was used to detect interstitial fibrosis. In addition, hematoxylin–eosin staining (Merck KGaA, Darmstadt, Hesse, Germany) was also carried out to detect tubular injury (blebbing of the apical membrane into the tubular lumen, cell fragments within the tubular lumen, flattening of the tubular epithelium, or loss of nuclei), tubular atrophy, and interstitial infiltrate of inflammatory cells [44 ]. Hematoxylin–eosin and Masson’s trichrome staining were performed according to standard procedures. Slices were evaluated via images captured with a Moticam 1080 digital camera (Motic China Group Co Ltd, Xiamen, Fujian, China) attached to a Moticam BA310E optical microscope (Motic China Group Co Ltd, Xiamen, Fujian, China) with 10× and 40× objectives. All images were captured under the same conditions of light and exposure.
Renal histopathologic lesions were calculated as the percentage of the total area observed under the microscope [45 (link)]. The total area of the renal tissue cut was considered 100%, and the percentage of the cortical area affected by tubular injury, interstitial fibrosis, tubular atrophy, and interstitial infiltration of inflammatory cells was quantified. Additionally, the total percentage of the renal cortex tissue altered histopathologically was calculated and was the result of the sum of the percentages of tubular injury, tubular atrophy, fibrosis, and inflammatory infiltrate in the renal cortex. Tubular injury was defined as the flattening of the tubular epithelium with calcified or noncalcified cellular fragments within their lumens, blebbing of the apical membrane into the tubular lumen, or loss of nuclei [44 ]. Interstitial fibrosis was defined as increased extracellular matrix separating tubules in the cortical area [46 (link)], demonstrated as the blue-stained areas on Masson’s trichrome stains [47 (link)]. Tubular atrophy was defined by thick, irregular tubular basement membranes, with decreased diameters of tubules [46 (link)]. Interstitial infiltrate of inflammatory cells was defined as an excess of inflammatory cells within the cortical interstitium [46 (link)]. The evaluations were carried out blindly by two anatomopathologic experts.
Renal histopathologic lesions were calculated as the percentage of the total area observed under the microscope [45 (link)]. The total area of the renal tissue cut was considered 100%, and the percentage of the cortical area affected by tubular injury, interstitial fibrosis, tubular atrophy, and interstitial infiltration of inflammatory cells was quantified. Additionally, the total percentage of the renal cortex tissue altered histopathologically was calculated and was the result of the sum of the percentages of tubular injury, tubular atrophy, fibrosis, and inflammatory infiltrate in the renal cortex. Tubular injury was defined as the flattening of the tubular epithelium with calcified or noncalcified cellular fragments within their lumens, blebbing of the apical membrane into the tubular lumen, or loss of nuclei [44 ]. Interstitial fibrosis was defined as increased extracellular matrix separating tubules in the cortical area [46 (link)], demonstrated as the blue-stained areas on Masson’s trichrome stains [47 (link)]. Tubular atrophy was defined by thick, irregular tubular basement membranes, with decreased diameters of tubules [46 (link)]. Interstitial infiltrate of inflammatory cells was defined as an excess of inflammatory cells within the cortical interstitium [46 (link)]. The evaluations were carried out blindly by two anatomopathologic experts.
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