Microvascular autophagy and caspase-3 activation are central regulators of renal fibrosis after ischemia-reperfusion
Hyunyun Kim, Francis Migneault, Shanshan Lan, Imane Kaci, Julie Turgeon, Annie Karakeussian Rimbaud, Martin Dupont, Shijie Qi, Mélanie Dieudé, Marie-Josée Hébert
Hyunyun Kim, Francis Migneault, Shanshan Lan, Imane Kaci, Julie Turgeon, Annie Karakeussian Rimbaud, Martin Dupont, Shijie Qi, Mélanie Dieudé, Marie-Josée Hébert
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Research Article Nephrology Vascular biology

Microvascular autophagy and caspase-3 activation are central regulators of renal fibrosis after ischemia-reperfusion

Abstract

Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) leading to renal fibrosis. Here, we investigate the kinetics of autophagy, apoptosis, and necroptosis activation in tubular epithelial cells (TECs) and peritubular capillaries (PTCs) after renal IRI, and their relative contributions to renal fibrogenesis. IRI with renal artery clamping in GFP-LC3 transgenic mice induced a predominant and sustained necroptotic response in TECs, while apoptosis and autophagy played minor roles. PTCs showed early and persistent activation of apoptosis, brief necroptosis induction, and increased autophagy at a distance from IRI. Disruption of the autophagic process with chloroquine (CHQ) injections in association with renal IRI did not modulate tubular death but enhanced PTC apoptosis and increased microvascular rarefaction and fibrosis. Apoptosis-deficient GFP-LC3/Caspase-3–/– mice exposed to renal IRI showed enhanced PTC autophagy, reduced PTC rarefaction, and inhibition of renal fibrosis, in spite of increased necroptosis in TECs. Inhibition of both autophagy with CHQ and apoptosis in GFP-LC3/Caspase-3–/– mice led to a marked switch toward necroptosis in PTCs. This was associated with aggravated microvascular rarefaction, increased leukocyte infiltration, and enhanced renal fibrosis. These findings establish a predominant role for PTC autophagy and caspase-3–dependent apoptosis in the development of renal fibrosis after IRI.

Authors

Hyunyun Kim, Francis Migneault, Shanshan Lan, Imane Kaci, Julie Turgeon, Annie Karakeussian Rimbaud, Martin Dupont, Shijie Qi, Mélanie Dieudé, Marie-Josée Hébert

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Figure 1

Renal IRI induces kidney dysfunction, tubular damage, microvascular rarefaction, and fibrosis.

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Renal IRI induces kidney dysfunction, tubular damage, microvascular rare...
(A) Serum creatinine and BUN levels up to 21 days after renal IRI (n = 3–5). IR30min, 30-minute ischemia/reperfusion. (B) Mean tubular injury scores in H&E-stained kidney sections (n = 4–5). The black arrow indicates injured tubules. (C) Mean number of peritubular capillaries (PTCs) with evidence of congestion in H&E-stained kidney sections (n = 4–5). The white arrow indicates PTC congestion. (D) Quantification of PLVAP immunohistochemistry (IHC) (n = 4–5). (E) Quantification of ACTA2 IHC (n = 5). The black arrow indicates ACTA2+ cells. (F) Quantification of Sirius red staining (n = 5). The white arrow indicates fibrosis. Quantification was performed with samples at baseline, 1, 2, 7, and 21 days after IRI. For each kidney, 10 randomly selected high-power fields (HPFs) (original magnification, ×200) were evaluated, consisting of 5 fields from the cortex and 5 from the corticomedullary junction. All scale bars: 50 μm. Values are mean ± SEM. P values obtained by 1-way ANOVA with Bonferroni’s post hoc test (A–C) or by unpaired, 2-tailed Student’s t test (D–F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for comparisons between baseline and each time point.