Prunetin in a GPR30-dependent manner mitigates renal ischemia/reperfusion injury in rats via interrupting indoxyl sulfate/TLR4/TRIF, RIPK1/RIPK3/MLKL, and RIPK3/PGAM5/DRP-1 crosstalk

Figures

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  Fig. 1. The impact of different doses of prunetin on renal function biomarkers (A) BUN and (B) Creatinine, as well as (C) kidney index in rats with I/R. Rats underwent renal I/R and then received different doses of prunetin (0.25, 0.5, and 1 mg/kg, i.p.) once daily for three successive days. Data are displayed as mean ± SD (n = 4) and were statistically analyzed by one-way ANOVA followed by Tukey's post-hoc test (P < 0.05). *, #, &, or % denote comparisons to SO, I/R, Pru 0.25 mg/kg, or Pru 0.5 mg/kg-treated groups, respectively. BUN: Blood urea nitrogen; I/R: Ischemia/reperfusion; Pru: Prunetin; SO: Sham-operated.

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  Fig. 2. Prunetin increased the (A) mRNA expression and (B) content of renal GPR30 in rats with I/R. Rats underwent renal I/R and then received a daily intraperitoneal injection of 1 mg/kg prunetin for three successive days alone or preceded by the GPR30 blocker, G-15. Data are displayed as mean ± SD (n = 4/6) and were statistically analyzed using one-way ANOVA followed by Tukey’s post-hoc test (P < 0.05). *, #, or @ denote comparisons to SO, I/R, or I/R + Pru-treated groups, respectively. GPR30: G protein-coupled receptor 30; I/R: Ischemia/reperfusion; Pru: Prunetin; SO: Sham-operated.

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  Fig. 3. Prunetin reduced serum level of (A) indoxyl sulfate and renal contents of (B) TLR4 and (C) TRIF in a GPR30-dependent manner in rats with I/R. Rats underwent renal I/R and then received a daily intraperitoneal injection of 1 mg/kg prunetin for three successive days alone or preceded by the GPR30 blocker, G-15. Data are displayed as mean ± SD (n = 6) and were statistically analyzed using one-way ANOVA followed by Tukey’s post-hoc test (P < 0.05). *, #, or @ denote comparisons to SO, I/R, or I/R + Pru-treated groups, respectively. I/R: Ischemia/reperfusion; Pru: Prunetin; SO: Sham-operated; TLR4: Toll-like receptor 4; TRIF: toll/interleukin-1 receptor-domain-containing adaptor inducing interferon-beta.

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  Fig. 4. Prunetin downregulated the protein expression of (A) p-RIPK1, (B) p-RIPK3, and (C) p-MLKL but upregulated that of (D) cleaved caspase-8 in rats with I/R. Rats underwent renal I/R and then received a daily intraperitoneal injection of 1 mg/kg prunetin for three successive days alone or preceded by the GPR30 blocker, G-15. Data are displayed as mean ± SD (n = 6) and were statistically analyzed using one-way ANOVA followed by Tukey’s post-hoc test (P < 0.05). *, #, or @ denote comparisons to SO, I/R, or I/R + Pru-treated groups, respectively. I/R: Ischemia/reperfusion; MLKL: Mixed lineage kinase domain-like; p: Phosphorylated; Pru: Prunetin; RIPK1/3: Receptor-interacting protein kinase 1/3; SO: Sham-operated; T: total.

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  Fig. 5. Prunetin amended mitochondrial fission by downregulating the protein expression of (A) PGAM5 and (B) p-DRP-1 in rats with I/R. Rats underwent renal I/R and then received a daily intraperitoneal injection of 1 mg/kg prunetin for three successive days alone or preceded with the GPR30 blocker, G-15. Data are displayed as mean ± SD (n = 6) and were statistically analyzed using one-way ANOVA followed by Tukey’s post-hoc test (P < 0.05). *, #, or @ denote comparisons to SO, I/R, or I/R + Pru-treated groups, respectively. DRP-1: Dynamin-related protein 1; I/R: Ischemia/reperfusion; p: Phosphorylated; PGAM5: Phosphoglycerate mutase 5; Pru: Prunetin; SO: Sham-operated; T: total.

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  Fig. 6. Prunetin restored the altered histological structure of the kidney. Photomicrographs representing H&E-stained renal tissue sections of rats (scale bar 50 μm). Sections of the (A) SO and the (B) SO + Pru reveal the regular histological structure of renal parenchyma (normal glomeruli [GL] and renal tubules [RT]). Section of the (C) I/R group depicts marked changes in renal tubules manifested by epithelial lining vacuolar degeneration [VD], eosinophilic renal cast [RC] in the lumen, and inflammatory cell infiltration [IF]. Section of the (D) Pru-treated group reveals slight VD of some renal tubules, whereas the section of the (E) G-15 blocker group + Pru shows congestion of renal blood vessels [CO], marked VD, coagulative necrosis of renal tubular epithelium [NC], RC in the lumen of renal tubules, and IF. These data are recapitulated in panel (F), which illustrates the total renal damage score in the different groups. The median (min–max) of 6 non-overlapping microscopic fields per animal (n = 3/group) was used to represent the non-parametric data, which were analyzed by the Kruskal-Wallis test followed by Dunn’s post-hoc test (P < 0.05). *, #, or @ denote comparisons to SO, I/R, or…

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  Fig. 7. Schematic presentation depicting molecular cascades involved in the reno-therapeutic effect of prunetin against I/R injury. Prunetin by activating GPR30 inhibits (1) the indoxyl sulfate/TLR4/TRIF inflammatory hub, (2) RIPK1/RIPK3/MLKL necroptotic trajectory, and (3) RIPK3/PGAM5/DRP-1 cue responsible for mitochondrial fission in the I/R kidney. DRP-1: Dynamin-related protein 1; GPR30: G protein-coupled receptor 30; I/R: Ischemia/Reperfusion; MLKL: Mixed lineage kinase domain-like; p: Phosphorylated; PGAM5: Phosphoglycerate mutase 5; RIPK1/3: Receptor-interacting protein kinase 1/3; TLR4: Toll-like receptor 4; TRIF: toll/interleukin-1 receptor-domain-containing adaptor inducing interferon-beta. This Fig. is created with BioRender.com.