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. 2021 Jul 1;12:683098. doi: 10.3389/fphys.2021.683098

Figure 5.

Figure 5

ANXA1sp treatment upregulates levels of sirtuin3 (SIRT3) in the mitochondria of kidney tubules. Mice were treated with either Vehicle or ANXA1sp 1 h prior to ischemia, subjected to 33 min of unilateral ischemia and contralateral nephrectomy, and then re-injected with Vehicle or ANXA1sp 1 h after reperfusion. Kidney tissues were harvested at 24 h after reperfusion. (A) mRNA levels of SIRT3 mRNA were determined by RT-PCR. Graph displays mean +/-SEM of SIRT3 normalized to GADPH, then normalized to Sham, Vehicle group (n = 3 sample for Sham groups, n = 12 samples for I/R groups). Statistical significance determined by two-way ANOVA with Sidak post-test (**p < 0.01). (B,C) Protein levels of SIRT3 were determined by Western blot. Representative western blot in (B) with graph in (C) displaying mean +/-SEM of densitometry of SIRT3 normalized to total protein (Tot. Prot.) from stain-free gel for each sample, then normalized to Sham, Vehicle group (n = 3 samples for Sham groups, n = 6–7 for I/R groups). Statistical significance determined by two-way ANOVA with Sidak post-test (*p < 0.05). (D) Paraffin-embedded kidney tissue was analyzed for SIRT3 (top) and mitochondrial complex IV (middle) expression by immunofluorescence microscopy with merged image (bottom). SIRT3 co-localizes with mitochondrial marker complex IV in kidney tubule cells. Scale bar shows 50 μm.