IRAP deficiency preserves renal function and attenuates pathology in aged mice

Figures

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  Figure 1. IRAP deficiency alleviates cellular senescence and prevents decline in renal function in ageing male mice (A) Mean arterial pressure, (B) GFR, (C) albuminuria, (D) glomerulosclerosis index (GSI), (E) renal interstitial fibrosis quantified via Masson’s trichrome staining, (F) perivascular fibrosis index (ratio of perivascular cross-sectional area to lumen cross-sectional area), (G) renal IRAP immunostaining positivity, (H) renal SA-β-gal, (I) nuclear p21 positivity (+), expressed as a proportion of total nuclei, and (J) urinary p21 excretion in male WT (red) and IRAP KO (blue) mice from 3 to 23 months of age. (K) mRNA expression of p21 (Cdkn1a), p16INK4a (Cdkn2a), and p53 (Trp53), expressed relative to the endogenous control (18s). (L) Histology panel shows PAS staining to determine GSI, Masson’s trichrome staining to detect tubulointerstitial fibrosis, SA-β-gal staining to detect senescent cells (blue staining), and p21 immunostaining (arrows point to p21+ nuclei). Scale = 50 μm. n = 4–10/group. (A–J) Data were analysed via two-way ANOVA followed by a Sidak’s multiple comparisons test; **P<0.01, ****P<0.0001 versus 3-month-old WT; †P<0.05, ††P<0.01, †††P<0.001 versus age-matched…

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  Figure 2. IRAP inhibition alleviates kidney fibrosis and improves kidney function in aged mice (A) GFR in male WT mice at 27 months of age, before and after 28 days of vehicle (purple) or HFI-419 (orange) infusion. (B) Albuminuria, (C) GSI, (D) renal interstitial fibrosis quantified via Masson’s trichrome staining, (E) perivascular fibrosis index (ratio of perivascular cross-sectional area to lumen cross-sectional area), (F) renal hydroxyproline content, (G) nuclear p21 positivity (+), expressed as a proportion of total nuclei, (H) urinary p21 excretion, and (I) renal mRNA expression of p21 (Cdkn1a), p16INK4a (Cdkn2a), and p53 (Trp53). A 3-month-old WT group is shown as a control (open points). (J) Histology panel shows PAS staining to determine GSI, Masson’s trichrome staining to detect tubulointerstitial and perivascular fibrosis, SA-β-gal staining to detect senescent cells (blue staining), and p21 immunostaining (arrows point to representative p21-positive cell nuclei). Scale = 50 μm. n = 4–10/group. (A) Data were analysed via two-way ANOVA followed by a Sidak’s multiple comparisons test; **P<0.01 versus 27-month-old vehicle; ††P<0.01 versus 28-month-old vehicle. Age effects were…

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  Figure 3. IRAP inhibition attenuates hydrogen peroxide-induced senescence and promotes wound healing in HK-2 cells HK-2 cells were exposed to DMSO (control), HFI-419 (0.01 μM), H2O2 (800 μM), or H2O2 ± HFI-419 for 24 h. (A) Cell viability measured via an MTS assay. (B) SA-β-gal+ cells (%) quantified using SA-β-gal staining (D, scale = 100 μm). (C) Wound closure (%) over 48 h, with representative images at the 48-h timepoint shown in (D; scale = 100 μm). Values represent mean ± SEM; n = 3 from independent experiments, each performed with three technical replicates. Data analysed via one-way ANOVA with Sidak’s multiple comparisons tests. Pairwise comparisons were control versus H2O2, control versus HFI-419, H2O2 versus HFI-419 and control versus HFI-419+ H2O2; **P<0.01, ***P<0.001, ****P<0.0001.

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  Figure 4. IRAP inhibition attenuates hydrogen peroxide-induced senescence and promotes wound healing in MDCK cells MDCK cells were exposed to DMSO (control), HFI-419 (0.01 μM), H2O2 (800 μM), or H2O2 ± HFI-419 for 24 h. (A) Cell viability measured via an MTS assay. (B) SA-β-gal+ cells (%) quantified using SA-β-gal staining (D, scale = 100 μm). (C) Wound closure (%) over 48 h, with representative images at the 48-h timepoint shown in (D; scale = 100 μm). Values represent mean ± SEM; n = 3 from independent experiments, each performed with three technical replicates. Data analysed via one-way ANOVA with Sidak’s multiple comparisons tests. Pairwise comparisons were control versus H2O2, control versus HFI-419, H2O2 versus HFI-419 and control versus HFI-419+ H2O2; *P<0.05, ***P<0.001, ****P<0.0001.

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  Figure 5. HFI-419 prevents H2O2-induced oxidative stress in HK-2 cells Metabolic profiling and ATP production rate of HK-2 cells treated with H2O2 (1.3 M) ± HFI-419 (0.01 μM) for 4 h. OCR presented as (A) the kinetic profile, (B) mean basal respiration, (C) mean proton leak, and (D) mean non-mitochondrial respiration. ECAR presented as (E) a kinetic profile, (F) mean non-glycolytic acidification, and (G) maximum glycolytic capacity. (H) Mean glycoATP production rates, (I) mean mitoATP production rates, (J) relative proportion of ATP production attributed to mitoATP and glycoATP, and (K) energetic map indicating shifts in metabolic phenotypes after treatment. Data are mean ± SEM; n = 3 from independent experiments, each performed with three technical replicates. (A,E,J,K) Data analysed via two-way ANOVA followed by Sidak’s multiple comparisons test. For panels (A,E), treatment groups were compared within each timepoint. For panels (J,K), P-values represent the main effects of H2O2 and HFI-419; *P<0.05, ***P<0.001. (B–D,F–I) Data analysed via one-way ANOVA with Sidak’s multiple comparisons tests. Pairwise comparisons were control versus H2O2, control versus HFI-419, H2O2 versus HFI-419 and…

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  Figure 6. IRAP inhibition has no effect on the metabolic profile of MDCK cells Metabolic profiling and ATP production rate of MDCK cells treated with H2O2 (1.3 M) ± HFI-419 (0.01 μM) for 4 h. OCR presented as (A) the kinetic profile, (B) mean basal respiration, (C) mean proton leak, and (D) mean non-mitochondrial respiration. ECAR presented as (E) a kinetic profile, (F) mean non-glycolytic acidification, and (G) maximum glycolytic capacity. (H) Mean glycoATP production rates, (I) mean mitoATP production rates, (J) relative proportion of ATP production attributed to mitoATP and glycoATP, and (K) energetic map indicating shifts in metabolic phenotypes after treatment. Data are mean ± SEM; n = 3 from independent experiments, each performed with three technical replicates. (A,E,J,K) Data analysed via two-way ANOVA followed by Sidak’s multiple comparisons test; For panels (A,E), treatment groups were compared within each timepoint, and for panels (J,K), no significant main effects of H2O2 and HFI-419 were detected. (B–D,F–I). Data analysed via one-way ANOVA, with no significant effects detected. Rot/AA, rotenone/antimycin A.`