Novel protective role of factor XII in kidney ischemia/reperfusion injury in a rat model Free

Renal ischemia/reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI) that often leads to rapid and severe renal dysfunction. IRI triggers a cascade of pathological sequelae including activation of multiple innate and adaptive immune cell compartments, generation of reactive oxygen species, upregulation of immune mediators, and complement activation. Together, these events lead to impairment of renal function. While there have been significant advancements in our understanding of the pathobiology of IRI, the precise mechanisms that drive or mitigate IRI-associated renal injury remain elusive. The contact pathway protease, factor XII (FXII), bridges activation of coagulation and key inflammatory pathways in numerous experimental settings. Thus, FXII has emerged as a promising therapeutic target. Given the potential of FXII to regulate inflammatory pathways important in renal IRI, we hypothesized that FXII promotes inopportune inflammatory responses in this context. To test this hypothesis, we induced bilateral renal IRI in gene-targeted FXII-deficient rats (FXII^-/-) and wild-type rats (WT) by cross clamping the renal arteries of both kidneys for 30 minutes, and allowed kidneys to reperfuse for 24 hours. Surprisingly, FXII^-/- rats exhibited higher levels of established markers or renal injury relative to WT rats following bilateral IRI. FXII^-/- animals had ~2-fold higher serum creatinine levels, and significantly higher levels of serum NGAL (Neutrophil Gelatinase-Associated Lipocalin), a marker of renal tubular injury. We also observed significantly higher levels of another marker of renal tubular damage, kidney injury molecule-1 (Kim-1), in kidney tissue from IRI-challenged FXII^-/- compared to IRI-challenged WT rats. To corroborate these biochemical findings, H&E-stained renal tissue sections from IRI challenged and unchallenged WT and FXII^-/- rats were systematically reviewed by a pathologist and scored using a semiquantitative multiparameter assessment tool. Kidneys from unchallenged rats appeared normal and indistinguishable between genotypes. Kidneys from IRI-challenged FXII^-/- rats had significantly higher disease scores relative to IRI-challenged WT rats that was driven primarily by differences in acute tubular necrosis. Consistent with more severe tubular injury, kidneys from FXII^-/- rats also demonstrated an almost 4-fold increase in apoptosis in renal tubular cells following IRI based on quantitation of TUNEL staining. Scant apoptotic cells were observed in unchallenged kidneys regardless of genotype. We also observed significantly increased macrophage infiltration, and increased markers of fibrosis (aSMA and collagen deposition) in IRI-challenged FXII^-/- kidneys. To begin to elucidate the mechanisms behind the more severe kidney injury observed in FXII^-/- rats we performed mass spectrometry-based proteomic analysis (LC-MS) on kidney tissues from IRI-challenged and unchallenged WT and FXII⁻^/⁻ rats and observed downregulation of several protein involved in cellular antioxidant capacity in FXII⁻^/⁻ compared to WT rats. Notably, electron microscopy (EM) analyses of kidney tissues from IRI challenged WT rats revealed modest evidence of damage, including focal loss of the brush border in tubular epithelial cells but normal mitochondria. In contrast, kidneys from IRI-challenged FXII^-/- rats show prominent mitochondrial damage, including vacuolar changes and breakdown of the cristae in mitochondria. Consistent with the renal tubular cells being the primary target of the more severe damage, glomeruli appeared intact in IRI-challenged WT and FXII^-/- renal tissue based on both H&E and EM analyses. Overall, these findings indicate that FXII deficiency is associated with exacerbated renal dysfunction after IRI-induced AKI. These findings reveal a novel and critical role of FXII in protecting the kidney from ischemia-reperfusion injury. Further mechanistic studies are warranted to determine the precise mechanisms by which FXII confers kidney protection, as well as understanding how this novel role for FXII could impact its potential as a clinical antithrombotic target.