Protective role of 17β-estradiol treatment in renal injury on female rats submitted to brain death

Figures

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  Figure 1. Experimental timeline. Sham, false-operated rats; BD, rats submitted to BD; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. BD, brain death.

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  Figure 2. Mean arterial pressure (A), serum (B) creatinine and (C) urea levels. Sham, false-operated rats; BD, rats submitted to BD; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Values represent the mean ± SEM (n=7). Statistical analysis: ANOVA (A) P=0.019; (B) PGroups=0.114 and PTime=0.025 (C) PGroups=0.955 and PTime<0.0001 (GraphPad Prism software v.9.1). *, P<0.05. BD, brain death; ANOVA, analysis of variance.

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  Figure 3. Analysis of leukocyte infiltration in renal tissue. (A) Leukocyte infiltration and (B) MPO stained cells. Sham, false-operated rats; BD, rats submitted to BD; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. (A) Formaldehyde-fixed sections of renal tissue were stained with hematoxylin and eosin. (B) Immunohistochemical analysis of MPO expression in kidney sections. Original magnification images: 20× for all images. Values represent the median and 95% percentile interval (n=6 per group; 1 tissue section per animal, 10 fields). Statistical analysis: Kruskal-Wallis (A) P=0.0639 and (B) P=0.018 (GraphPad Prism software v.9.1). MPO, myeloperoxidase; BD, brain death.

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  Figure 4. Protein (A) and gene expression (B) of KIM-1 in renal tissue. Sham, false-operated rats; BD, rats submitted to BD; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Values represent the median and 95% percentile interval. (A) Imunohistochemical analysis 5 photos/sample, 1 sample/rat and 6 rats/group. (B) Gene expression levels are relative to those in naïve rats. Statistical analysis: Kruskal-Wallis (A) P<0.0001 and (B) P=0.078 (GraphPad Prism software v.9.1). KIM, kidney injury marker; BD, brain death.

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  Figure 5. Protein and gene expression of Caspase-3 (A,C) and BCL-2 (B,D). Sham, false-operated rats; BD, rats submitted to BD; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Values represent the median and 95% percentile interval. (A,B) Imunohistochemical analysis 5 photos/sample and 2 samples/animal. (C,D) Gene expression levels are relative to those in naïve rats in 5 animals/group. Statistical analysis: Kruskal-Wallis (A) P=0.188, (B) P=0.561, (C) P=0.016 and (D) P=0.199 (GraphPad Prism software v.9.1). BD, brain death.

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  Figure 6. Quantification of C3 in serum (A); protein (B) and gene expression (C); membrane attack complex (MAC/C5b-9) protein expression (D) in renal tissue. Sham, false-operated rats; BD, rats submitted to BD; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Values represent the median and 95% percentile interval. (B,D) Imunohistochemical analysis 5 photos/sample, 1 sample/rat and 6 rats/group. (C) Gene expression levels are relative to those in naïve rats. Statistical analysis: Kruskal-Wallis (A) P=0.112, (B) P=0.004, (C) P=0.827 and (D) P=0.0004 (GraphPad Prism software v.9.1). BD, brain death.

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  Figure 7. Gene expression of complement receptor for C3a (C3aR) (A) and C5a (C5aR) (B), DAF and MAC-IP in renal tissue. Sham, false-operated rats; BD, rats submitted to BD; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Values represent the median and 95% percentile interval for 1 sample/rat and 6 rats/group. Gene expression levels are relative to those in naïve rats. Statistical analysis: Kruskal-Wallis (A) P=0.054, (B) P=0.019, (C) P=0.281 and (D) P=0.361 (GraphPad Prism software v.9.1). BD, brain death; DAF, decay-accelerating factor; MAC-IP, membrane attack complex-inhibitor protein.

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  Figure 8. Protein and expression of metalloproteinase 2 (MMP2) (A,C) and metalloproteinase 9 (MMP9) (B,D). Sham, false-operated rats; BD, rats submitted to BD; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Values represent the median and 95% percentile interval. (A,B) Imunohistochemical analysis 5 photos/sample and 2 samples/animal. (C,D) Gene expression levels are relative to those in naïve rats in 5 animals/group. Statistical analysis: Kruskal-Wallis (A) P=0.043, (B) P=0.011, (C) P=0.924 and (D) P=0.063 (GraphPad Prism software v.9.1).

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  Figure 9. Protein and gene expression of IL-1β (A,D), IL-6 (B,E), and VEGF (C,F) in renal tissue. Sham, false-operated rats; BD, rats submitted to BD; E2-T0, 17β-estradiol (E2)-treated rats after confirmation of BD and E2-T3, 17β-estradiol (E2)-treated rats after 3 h of confirmation of BD. Values represent the median and 95% percentile interval for 1 sample/rat and 6 rats/group. Gene expression levels are relative to those in naïve rats. Statistical analysis: Kruskal-Wallis (A) P=0.129, (B) P=0.024, (C) P=0.011, (D) P=0.030, (E) P=0.004 and (F) P=0.349 (GraphPad Prism software v.9.1).