Kidney nitrosative stress response to acute ischemia‐reperfusion injury is present only in the external medulla and independent of sex in adult rats

Abstract

Despite the morphological and physiological differences between male and female kidneys., and the differences in the pathophysiological response to ischemia-reperfusion injury at different experimental models (genetic, subcellular, cellular, animals and humans), they're still controversies between if these responses are depended to nitric oxide - nitrosative stress vía, and the site in the renal parenchyma which are predominant. METHODS We analyzed (Image G program and Wilcoxon test in JMP software) the level of nitrosylated protein (fluorescent microscopy, DHPEF-filter, 20x objective, 0.5 slight exposure, antibodies AB61392, revealed with antibody-CF488A, diluted 1:1000) in 10-µm slices of left-kidney subjected to 45 min ischemia and 24 h reperfusion, both male (n=5 w= 120-150g) and female Wistar adult rats (n=9, w=120-150g). The right kidney of the same animals was the control kidney (no ischemia). RESULTS The level of nitrosylated protein were similar between groups for the internal medulla (ischemic kidney male= 115.24±10.73, ischemic kidney female= 130.97 ± 2.54, control kidney male= 107.72 ± 3.41, control kidney female= 112.53 ± 3.31, p=ns) and the glomeruli (ischemic kidney male= 122.88 ± 16.13, ischemic kidney female= 117.79 ± 9.26, control kidney male= 101.58 ± 13.75, control kidney female= 98.26 ± 5.75, p=ns). For the external medulla, there was an increase in the level of nitrosylated protein after the treatment (45 min ischemia, 24 h reperfusion), in both male and females (ischemic kidney male= 121.81 ± 10.97, ischemic kidney female= 125.96 ± 3.19) compared with control kidney (male= 93.75 ± 4.94, female= 99.78 ± 2.29, p=0.0004 ischemic vs control female kidney, p=0.0069 ischemic vs controlled male kidney, p=0.01 ischemic male vs control female kidney), but without differences between sexes. CONCLUSION The level of nitrosative protein was similar in control kidney (without ischemia) in both sexes, which represents a nitrosative basal level necessary for physiological responses and independent of the protein level sex-differences reported in the literature (SOD, eNOS and iNOS, among others). Also, the level of nitrosative protein was increased in 29% in males and 26% in females only in the external medulla, without differences in the other sections of renal parenchyma. The results of this work showed similarities in epigenetic changes secondary to flow interruption of the whole renal pedicle in female and male rats only in the external medulla, but not a behavior pattern in the papilla or cortex. In-situ study of this phenomenon, in contrast to other techniques that require tissue homogenization (like flow cytometry or western blot), allowed for a precise quantitative signal measurement in each region.