*Purpose: Ischemia-reperfusion injury (IRI) is a major risk factor for allograft dysfunction and chronic renal failure. Although tubular epithelial cells (TEC) are considered the main cellular target, evidence identifies microvascular injury as an important contributor. Caspase-3 activation, a read-out of apoptotic cell death, has been reported in TEC and endothelial cells in acute kidney injury (AKI). The functional and importance of caspase-3 activation in tubular and microvascular demise post-IRI remains ill defined. 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 KO mice. HUVEC (human umbilical vascular endothelial cell) and PT2 TECs (human proximal renal tubular cells) cultures are exposed to hypoxia-reoxygenation treatment to mimic IRI in vivo.

*Results: In vitro, we found that caspase-3 silencing significantly decreased apoptosis in endothelial cells (HUVEC) but increased necrosis in tubular epithelial cells sumitted to hypoxia-reoxygenation treatment. In murine model of IRI, we found that compared with their wild-type counterparts, caspase-3 KO mice in the early stage of AKI had high urinary cystatin C levels, tubular injury scores, and serum creatinine levels. Electron microscopy revealed evidence of tubular epithelial cell necrosis in caspase-3 KO mice, and immunohistochemistry showed upregulation of the necroptosis marker receptor interacting protein kinase 3 (RIPK3) in renal cortical sections. Western blot analysis further demonstrated enhanced levels of phosphorylated RIPK3 in the kidneys of caspase-3 KO mice. In contrast, caspase-3 KO 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 KO mice had reduced microvascular rarefaction and renal fibrosis, as well as decreased expression of a-smooth muscle actin and reduced collagen deposition within peritubular capillaries. Moreover, caspase-3 KO mice exhibited signs of reduced tubular ischemia, including lower tubular expression of hypoxia-inducible factor-1a and improved tubular injury scores.

*Conclusions: 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.

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