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[Preprint]. 2023 Oct 3:2023.10.03.560700. [Version 1] doi: 10.1101/2023.10.03.560700

Figure 3. Post-ischemic simultaneous inactivation of endothelial PHD1, 2, and 3 promotes maladaptive kidney repair.

Figure 3.

(A) Scheme illustrating the experimental strategy applied for unilateral renal IRI (uIRI) studies. PHDTiEC mice and their Cre− littermates were subjected to 25 minutes of unilateral renal artery clamping. Treatment with tamoxifen was started on day 1 post uIRI involving 4 IP doses given every other day. Mice were sacrificed for histopathological and molecular analysis on day 14 post uIRI. (B) Representative images of H&E and Picro-Sirius red stained sections as well as tubular injury score and semi-quantitative analysis of Picro-Sirius red+ve area on day 14 post-ischemic kidneys from PHDTiEC mice and Cre− littermates. Scale bars indicate 100 μm and 200 μm for H&E and Picro-Sirius red images, respectively. (C) mRNA levels of Loxl2, Tgfb1 and Acta2 in IR and CTL kidneys from PHDTiEC mice or their Cre− controls at day 14 after uIRI. (D) Representative images of EMCN immunostaining and semiquantitative analysis of EMCN+ve peritubular capillary area on day 14 post-ischemic kidneys from PHDTiEC mice and Cre− littermates. (E) Scheme depicting the experimental workflow for bilateral renal IRI (bIRI) studies. PHDTiEC mice and their Cre− littermates were subjected to 23 minutes of bilateral renal artery clamping followed by tamoxifen treatment as described in A. Serum BUN levels were measured on 1 day prior to bIRI (baseline) and on days 1, 7 and 14 post bIRI. GFR was measured by FITC-sinistrin clearance on day 14 post bIRI using the MediBeacon transdermal GFR monitor system. (F) Serum BUN levels at different time points and GFR measurements on day 14 post bIRI. All bars show mean ± SEM of each group (n=4–8). For (B), (D) and (F), statistics were determined by unpaired t-test with Welch’s correction. For (C), one-way ANOVA with Sidak correction for multiple comparisons was used. *, P< 0.05; **, P< 0.01; ns, not statistically significant. CTL, contralateral kidney; IR, kidney subjected to IRI; bIRI, bilateral IRI; Rel., relative.