Endothelial HIF-2 mediates protection and recovery from ischemic kidney injury

Abstract

The hypoxia-inducible transcription factors HIF-1 and HIF-2 mediate key cellular adaptions to hypoxia and contribute to renal homeostasis and pathophysiology; however, little is known about the cell type-specific functions of HIF-1 and HIF-2 in response to ischemic kidney injury. Here, we used a genetic approach to specifically dissect the roles of endothelial HIF-1 and HIF-2 in murine models of hypoxic kidney injury induced by ischemia reperfusion or ureteral obstruction. In both models, inactivation of endothelial HIF increased injury-associated renal inflammation and fibrosis. Specifically, inactivation of endothelial HIF-2α, but not endothelial HIF-1α, resulted in increased expression of renal injury markers and inflammatory cell infiltration in the postischemic kidney, which was reversed by blockade of vascular cell adhesion molecule-1 (VCAM1) and very late antigen-4 (VLA4) using monoclonal antibodies. In contrast, pharmacologic or genetic activation of HIF via HIF prolyl-hydroxylase inhibition protected wild-type animals from ischemic kidney injury and inflammation; however, these same protective effects were not observed in HIF prolyl-hydroxylase inhibitor-treated animals lacking endothelial HIF-2. Taken together, our data indicate that endothelial HIF-2 protects from hypoxia-induced renal damage and represents a potential therapeutic target for renoprotection and prevention of fibrosis following acute ischemic injury.

Figure 1. Characterization of mice with EC-specific inactivation of HIF-1α and HIF-2α.

(A) Scheme illustrating the strategy used to assess the degree of EC-specific recombination. Cdh5-Cre mice were crossed to ROSA26-ACTB-tdTomato,-EGFP reporter (mT/mG) mice, and ECs were analyzed by FACS. Graph on the right shows the percentage of GFP-positive cells contained within the CD31-positive/CD45-negative cell population isolated from kidneys or lungs. (B) Left: representative images of CD31-stained cortex and medulla of kidneys from EC-specific Hif1aHif2a^–/– and Cre^– control mice. Right: quantification of CD31-positive area (n = 5). (C) Shown are representative images of cablin-stained peritubular capillaries in renal cortex and medulla imaged with confocal laser-scanning microscopy. The anti-cablin antibody used here preferentially stains the basal lamina of peritubular renal capillaries and does not label glomerular capillaries or small arterioles. (D) Quantification of baseline vascular permeability in kidney and lung tissue from 8-week-old mice using the EBD vascular permeability assay (n = 4–6). Graph bars represent mean values ± SEM; *P < 0.05. Scale bars: 100 μm.

Figure 2. Endothelial HIF protects from UUO-induced renal injury and inflammation.

(A) Shown are representative Sirius red–stained kidney sections and corresponding quantification of Sirius red–positive areas at days 8 and 12 following UUO (n = 10–12). (B) Representative images of CD31-stained renal cortex or medulla from mutant and control kidneys (day 12 after UUO); panels on right show quantification of CD31-positive areas in cortex or medulla (n = 10) and corresponding Cd31 mRNA levels in UUO (day 12) and CTL kidneys from Hif1aHif2a^–/– mice and controls. (C) Shown are F4/80-stained representative kidney sections from Hif1aHif2a^–/– mutants and Cre^– mice at day 12 following UUO (n = 10–11). Graph shows the number of F4/80-positive cells per hpf. Graph bars represent mean values ± SEM; *P < 0.05. UUO, kidney subjected to UUO; CTL, contralateral kidney. Scale bars: 100 μm.

Figure 3. Endothelial HIF promotes recovery from renal IRI.

(A) Schematic illustration of injury model and representative images of H&E-stained sections of injured kidneys from Hif1aHif2a^–/– mutants and Cre^– littermate controls at 2 hours, 1 day, and 3 days following unilateral IRI. Arrows point to necrotic tubules; asterisks indicate tubules with cast-forming material. (B) Left panel: time course analysis of Kim1 mRNA levels in injured kidneys. Right panel: mRNA levels of Kim1 in injured and contralateral kidneys at day 3 after IRI (n = 5–6). (C) BUN levels at day 1 and 3 following bilateral renal IRI in Hif1aHif2a^–/– and controls (n = 6–14). Graph bars represent mean values ± SEM; *P < 0.05. IR, kidney subjected to unilateral renal ischemia-reperfusion. Scale bars: 100 μm.

Figure 4. Inactivation of endothelial HIF promotes IRI-associated tubulointerstitial fibrosis.

(A) Representative images of Sirius red–stained kidneys from EC-specific Hif1aHif2a^–/– and Cre^– control mice at day 9 after IRI. Right panel shows quantification of Sirius red–positive area in cortex and medulla (n = 4). (B) RT-PCR analysis of Col18a1, Loxl-2, Tgfb1, and Col1a1 mRNA levels in mutant and control kidneys at day 9 following IRI (n = 4). (C and D) Representative images of CD31- and F4/80-stained kidneys analyzed on day 9 after renal clamping. Right panels: quantification of CD31-positive area and F4/80-positive cell number/hpf (n = 4–5). Graph bars represent mean values ± SEM; *P < 0.05; **P < 0.01. Scale bars: 100 μm.

Figure 5. Endothelial HIF suppresses IRI-associated renal inflammation.

(A) Representative images of CD45-stained sections of injured kidneys from Hif1aHif2a^–/– mutant mice and Cre^– littermate controls at 2 hours, 1 day, and 3 days following ischemia reperfusion. Bottom panel shows quantification of CD45-positive area in Hif1aHif2a^–/– and Cre^– controls (n = 6). (B) Transcript levels of Sele, Icam1, and Vcam1 in injured kidneys at time points indicated (n = 5–6). Graph bars represent mean values ± SEM. *P < 0.05; **P < 0.01. Scale bars: 100 μm.

Figure 6. Blockade of VCAM1/VLA4 reverses renal injury associated with endothelial HIF deletion.

Overview of the experimental protocol and representative images of H&E-stained kidney sections from control IgG- or anti-VCAM1/VLA4–treated Hif1aHif2a^–/– mutants (A) or Cre^– mice (B) on day 3 after IRI. Respective bottom panels show corresponding Kim1 mRNA levels (n = 6–7), MPO activity, and quantitative RT-PCR analysis for Vcam1 and Icam1 in IR and CTL kidneys on day 3 after IRI. Graph bars represent mean values ± SEM. *P < 0.05; **P < 0.01. Scale bars: 100 μm.

Figure 7. Inactivation of endothelial HIF-2α but not HIF-1α impairs recovery from kidney injury and inflammation.

(A) Representative images of CD45-stained sections of injured kidneys from EC-specific Hif1a^–/–, Hif2a^–/–, or Cre^– littermate control mice. Right panel shows percentage of CD45-positive area (n = 3–6). (B) Relative Vcam1 mRNA levels in IR and CTL kidneys on day 3 after IRI (n = 5–6). (C) Representative images of H&E-stained kidney sections 3 days after renal IRI in Hif1a^–/–, Hif2a^–/–, or Cre^– littermate controls. Arrow indicates a necrotic dilated tubule; asterisks mark tubules with cast-forming material. Kim1 mRNA levels in IR and CTL kidneys are shown on right (n = 6). Graph bars represent mean values ± SEM. *P < 0.05; **P < 0.01. Scale bars: 100 μm.

Figure 8. Inactivation of endothelial PHD2 attenuates renal IRI and suppresses the induction of EC-adhesion molecules.

(A) Representative images of H&E-stained kidney sections from Phd2^–/– or Cre^– littermate controls 3 days after renal IRI. Arrow depicts tubular necrosis. Asterisk indicates cast. Lower graphs show semiquantitative scores for dilated tubules and tubules with cast-forming material (n = 5–7). Kim1 mRNA levels in IR and CTL kidneys are shown on right (n = 5–7). (B) Relative Vcam1 and Icam1 mRNA levels in IR and CTL kidneys on day 3 after IRI (n = 5–7). (C) HUVECs were treated with DMOG or vehicle during hypoxia exposure and subjected to reoxygenation in the presence of TNF-α. Left panels: VCAM1 and ICAM1 mRNA levels in HUVECs. Right panel: functional adhesion assay with fluorochrome-labeled THP-1 cells. THP-1 cells were cocultured for 1 hour with HUVECs; the degree of adhesion is measured in relative fluorescence units. Graph bars represent mean values ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001. Scale bars: 100 μm. veh, vehicle (DMSO).

Figure 9. Renoprotection induced by systemic HIF prolyl-hydroxylase inhibition requires endothelial HIF-2.

(A) Scheme of the experimental protocol and representative images of H&E-stained kidney sections from vehicle- or PHI-treated WT animals on day 3 after IRI. Arrow indicates tubular necrosis. Hatch mark shows dilatation. Asterisk indicates casts. Bottom panel on the right shows corresponding Kim1 mRNA levels (n = 6). (B) Representative images of CD45-stained sections from injured kidneys of vehicle- or PHI-treated WT animals. Graph shows quantitative analysis of CD45-positive area (n = 6). (C) Relative levels of Vcam1 and Icam1 mRNA (n = 6). (D) Representative images of H&E-stained kidney sections from PHI- or vehicle-treated EC-specific Hif2a^–/– mutants and corresponding Kim1 mRNA levels (n = 4–5). Arrows depict tubular necrosis. Asterisks indicate casts. (E) Renal transcript levels of Vcam1 and Icam1 in PHI- or vehicle-treated Hif2a^–/– mice (n = 4–5). (F) Schematic depicting the role of the endothelial PHD/HIF-2 axis in renal ischemic injury. Graph bars represent mean values ± SEM. *P < 0.05; **P < 0.01. Scale bars: 100 μm. PHI, HIF prolyl-hydroxylase inhibitor; veh, vehicle (1% methylcellulose).