Caspase-3 Is a Pivotal Regulator of Microvascular Rarefaction and Renal Fibrosis after Ischemia-Reperfusion Injury

Abstract

Background Ischemia-reperfusion injury (IRI) is a major risk factor for chronic renal failure. Here, we characterize the different modes of programmed cell death in the tubular and microvascular compartments during the various stages of IRI-induced AKI, and their relative importance to renal fibrogenesis.Methods We performed unilateral renal artery clamping for 30 minutes and contralateral nephrectomy in wild-type mice (C57BL/6) or caspase-3^-/- mice.Results Compared with their wild-type counterparts, caspase-3^-/- mice in the early stage of AKI had high urine cystatin C levels, tubular injury scores, and serum creatinine levels. Electron microscopy revealed evidence of tubular epithelial cell necrosis in caspase-3^-/- mice, and immunohistochemistry showed upregulation of the necroptosis marker receptor-interacting serine/threonine-protein kinase 3 (RIPK3) in renal cortical sections. Western blot analysis further demonstrated enhanced levels of phosphorylated RIPK3 in the kidneys of caspase-3^-/- mice. In contrast, caspase-3^-/- mice had less microvascular congestion and activation in the early and extension phases of AKI. In the long term (3 weeks after IRI), caspase-3^-/- mice had reduced microvascular rarefaction and renal fibrosis, as well as decreased expression of α-smooth muscle actin and reduced collagen deposition within peritubular capillaries. Moreover, caspase-3^-/- mice exhibited signs of reduced tubular ischemia, including lower tubular expression of hypoxia-inducible factor-1α and improved tubular injury scores.Conclusions These results establish the pivotal importance of caspase-3 in regulating microvascular endothelial cell apoptosis and renal fibrosis after IRI. These findings also demonstrate the predominant role of microvascular over tubular injury as a driver of progressive renal damage and fibrosis after IRI.

Keywords: acute kidney injury; apoptosis; caspase-3; ischemia-reperfusion.

Figure 1.

Caspase-3 deficiency aggravates IRI-induced tubular injury. (A) Serum creatinine levels in wild-type (WT) and caspase-3^−/− (KO) mice at baseline (pre-op), and 1, 2, 3, and 7 days post-IRI. (B) Representative hematoxylin and eosin (H&E)–stained renal sections from WT and KO mice at 1, 2, 3, and 7 days post-IRI (original magnification ×200). (C) Left panel: mean tubular injury scores of ten randomly chosen high-power fields (original magnification ×200) in mice kidney sections post-IRI. Right panel: urinary levels of cystatin C in WT and KO mice at baseline (pre-op) and 1, 2, 3, or 7 days post-IRI. (D) Left panels: representative Kidney Injury Molecule 1 (KIM-1) immunohistochemistry in renal cortical sections from WT and KO mice at day 1 post-IRI (magnification 200×). Right panel: quantification of KIM-1 immunohistochemistry-stained murine renal cortical sections at baseline (pre-op) and 1 day post-IRI. All scale bars, 50 μm. Values are mean±SEM. *P<0.05, compared between WT and KO at the same time point.

Figure 2.

Caspase-3 deficiency increases IRI-induced necroptosis in tubular cells. (A) Representative electron microscopy (EM) showing renal tubules from wild-type (WT) (left) and caspase-3^−/− (KO) (right) mice that underwent IRI and were euthanized at 1 day post-IRI (original magnification ×1000). Scale bars, 10 μm. Tubules from KO mice show severe necrotic changes with loss or tubular cell membrane integrity and widespread accumulation of cellular debris within tubules. (B) Left panels: representative RIPK3 immunohistochemistry (IHC) in renal cortical sections from WT and KO mice that underwent IRI and were euthanized at 1 day post-IRI (original magnification ×200). Right panel: quantification of RIPK3 IHC in murine renal cortical sections at baseline (pre-op) and 1 day post-IRI. Scale bars, 50 μm. (C) Left panel: representative Western blot (WB) of phosphorylated RIPK3 (pRIPK3) in renal tissue from WT and KO mice that underwent IRI and were euthanized at 1 day post-IRI. Right panel: quantification of pRIPK3 WB at day 1 post-IRI. Values are mean±SEM. *P<0.05, compared between WT and KO at the same time point.

Figure 3.

Caspase-3 deficiency attenuates IRI-induced microvascular injury. (A) Left panel: quantification of rouleaux formation in hematoxylin and eosin (H&E)–stained kidney sections at baseline (pre-op) and from mice that underwent IRI and were euthanized at 1, 2, 3, or 7 days post-IRI. Right panel: representative H&E-stained murine renal cortical sections at 3 days post-IRI (original magnification ×400). Scale bars, 20 μm. (B): Left panel: representative images of MECA-32 immunohistochemistry (IHC) in renal cortical sections from wild-type (WT) and caspase-3^−/− (KO) mice that underwent IRI and were euthanized at 3 days post-IRI (original magnification ×200 and ×400). Right panel: quantification of MECA-32 in murine renal cortical medullary junction sections at baseline (pre-op) and 1, 2, 3, or 7 days post-IRI. Scale bars, 50 μm. (C) Representative electron microscopy images of renal endothelial cells in WT (left) and KO (right) mice that underwent IRI and were euthanized at 3 days post-IRI. Loss of endothelial fenestration is found in WT mice (arrow), whereas KO show preserved fenestrae (original magnification ×1000). Scale bars, 500 nm. Values are mean±SEM. *P<0.05, compared between WT and KO at the same time point.

Figure 4.

Caspase-3 deficiency attenuates IRI-induced upregulation of profibrotic markers. (A) Serum levels of CTGF in wild-type (WT) and caspase-3^−/− (KO) mice that underwent IRI and were euthanized at 1, 2, 3, or 7 days post-IRI. (B) Left panel: quantification of α-SMA immunohistochemistry (IHC) in renal cortical sections from WT and KO mice at baseline (pre) or at 1, 2, 3, or 7 days post-IRI (original magnification X200). Right panels: representative α-SMA staining in murine renal cortical sections at day 3 after surgery. Scale bars, 50 μm. (C) Left panels: representative images of Sirius Red staining in renal cortical medullary junction sections from WT (top panel) and KO (bottom panel) mice that underwent IRI and were euthanized at 7 days post-IRI (original magnification X200). Scale bars, 50 μm. Right panel: quantification of Sirius Red staining in murine renal cortical medullary junction sections at baseline and 1, 2, 3, or 7 days post-IRI. Values are mean±SEM. *P<0.05, compared between WT and KO at the same time point.

Figure 5.

Caspase-3 deficiency attenuates IRI-induced microvascular rarefaction and fibrosis. (A) Left panels: representative images of MECA-32 immunohistochemistry (IHC) in cortical medullary junction sections from wild-type (WT) and caspase-3^−/− (KO) mice that underwent IRI and were euthanized at 21 days post-IRI (original magnification ×200 and ×400). Right panel: quantification of MECA-32 staining in murine renal cortical sections at 21 days post-IRI. Scale bars, 50 μm. (B) Left panels: representative image of α-SMA IHC of renal cortical sections from WT and KO mice that underwent IRI and were euthanized at 21 days post-IRI (n=10 per group; original magnification X200 and X400). Right panel: quantification of α-SMA staining in PTCs in murine renal cortical sections at 21 days post-IRI. Scale bars, 50 μm. (C) Left panel: representative Western blot (WB) of α-SMA from WT and KO mice that underwent IRI and were euthanized at 21 days post-IRI. Right panel: quantification of α-SMA WB at 21 day post-IRI. (D) Left panels: representative image of Sirius Red staining of renal cortical medullary junction sections from WT and KO mice that underwent IRI and were euthanized at 21 days post-IRI (original magnification ×200 and ×400). Right panel: quantification of Sirius Red staining of murine renal cortical medullary junction sections at 21 days post-IRI. Scale bars, 50 μm. Values are mean±SEM. *P<0.05; ***P<0.001, compared between WT and KO at the same time point.

Figure 6.

Caspase-3 deficiency prevents IRI-induced long-term endothelial cell death. (A) Representative electronic microscopic images of endothelial cell apoptotic death from wild-type (WT) and caspase-3^−/− (KO) mice that underwent IRI and were euthanized at 21 days post-IRI. Red arrows indicate apoptotic bodies and blue arrows indicate exosome-like membrane vesicles (original magnification ×1000). Scale bars, 500 nm. (B) Quantification of apoptotic endothelial cells (ECs) in WT and KO mice that underwent IRI and were euthanized at 21 days post-IRI. Values are mean±SEM. *P<0.05, compared between WT and KO at the same time point.

Figure 7.

Caspase-3 deficiency prevents IRI-induced long-term tubular injury. (A) Left panels: representative images of HIF-1α staining in murine renal cortical sections 21 days after surgery. Right panel: quantification of HIF-1α immunohistochemistry (IHC) in renal cortical sections from wild-type (WT) and caspase-3^−/− (KO) mice at baseline and 21 days post-IRI (original magnification ×200). (B) Left panel: representative Western blot (WB) images of HIF-1α in renal tissue 21 days post-IRI. Right panel: quantification of HIF-1α WB detection at 21 days post-IRI. (C) Representative hematoxylin and eosin (H&E)–stained murine renal cortical sections 21 days post-IRI (original magnification ×200). (D) Mean tubular injury scores of ten randomly chosen high-power fields in wild-type (WT) and caspase-3^−/− (KO) mice that underwent IRI and were euthanized at 21 days post-IRI. (E) Serum creatinine levels in WT and KO mice that underwent IRI and were euthanized at 21 days post-IRI. All bar scales=50 μm. Values are mean±SEM. *P<0.05, compared between WT and KO at the same time point.

Figure 8.

Caspase-3 silencing decreases apoptosis in endothelial cells but increases necrosis in TECs submitted to hypoxia plus serum starvation and reoxygenation. (A) Left panels: representative images of Hoechst 33342 and propidium iodide (Ho-PI) staining in HUVECs exposed to hypoxia (5% O₂) for 4 hours, followed by reoxygenation for 1 hour in serum-free medium. Right panels: representative images of western blot (top panel) and quantification (bottom panel) for pro-caspase-3 in HUVECs transfected with siRNA control (si ctl) or caspase-3 (si casp3; n=3 independent experiments). (B) Left panels: representative images of Hoechst 33342 and propidium iodide (Ho-PI) staining of human PT-2 TECs exposed to hypoxia (5% O₂) for 4 or 24 hours followed by reoxygenation for 1 hour in serum-free medium. Right panels: representative images of Western blot and quantification for pro-caspase-3 in PT-2 cells transfected with si ctl or caspase-3 (n=3 independent experiments). (C) Left panel: quantification of apoptotic death in HUVECs and PT-2 cells exposed to hypoxia reoxygenation in serum-free medium. Right panel: quantification of necrotic death in HUVECs and PT-2 cells exposed to hypoxia reoxygenation in serum-free medium. Values are mean±SEM. *P<0.05.

Figure 9.

Schematic representation of the effect of caspase-3 deficiency on microvascular rarefaction and renal dysfunction post-IRI. Caspase-3 is a pivotal regulator of PTC rarefaction and renal dysfunction. Early tubular injury and renal dysfunction are increased in caspase-3^−/− (KO) mice, whereas microvascular integrity is ameliorated throughout the various phases. In the long term, KO mice show reduced microvascular dropout, decreased tubular injury, and reduced interstitial fibrosis. EC, endothelial cell; WT, wild type.