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 3

Renal endothelial cells exhibit a unique mRNA expression profile related to cell death compared with whole kidney lysates.

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Renal endothelial cells exhibit a unique mRNA expression profile related...
(A) Heatmap illustrating the expression of genes involved in apoptosis, necroptosis, and autophagy (derived from the KEGG and Reactome pathways) in renal endothelial cells and kidney lysate groups; n = 4. (B) Biological process enrichment analysis of transcripts differentially expressed in renal endothelial cells compared with the whole kidney lysate. An FDR-adjusted P value of less than 0.05 was considered significant. (C) Volcano plot of transcripts identified in the biological process “Positive regulation of apoptotic process” and enriched in kidney endothelial cells (EC). The plot displays the significantly differentially expressed transcripts identified through transcriptomic analysis. Transcripts are ranked in the volcano plot according to their FDR-adjusted P value (y axis) as –log10 and their relative enrichment ratio (log2) between endothelial cells and whole kidney lysate (x axis). Red dots represent the transcripts selected for validation by quantitative RT-PCR. (D) CD31+ and CD31– cells were isolated from whole kidney. The expression of CD31, Epcam, Fas, Caspase-8, and Caspase-12 mRNAs was measured by quantitative RT-PCR. The results are presented as the relative expression of mRNA ± SEM after normalization to Hprt1; n = 3 for each group. P values were obtained by unpaired, 2-tailed Student’s t test. *P < 0.05, **P < 0.01.