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  • br Materials and methods br Results br Discussion AKI


    Materials and methods
    Discussion AKI is a frequent complication in ICU patients, and approximately 50% of AKI cases are attributable to sepsis [21]. Sepsis and AKI synergistically increase morbidity and mortality in ICU patients. Therefore, a more complete molecular understanding of renal tubular damage has become an area of exploration for the development of novel therapies for AKI. In the present study, we demonstrate that TIMP2 together with inflammatory cytokines are increased both in vitro and in vivo in response to LPS and sepsis, respectively. These molecules induced apoptosis, decreased cell viability in vitro, and compromised renal function as measured by the serum creatinine level in CLP-induced sepsis in mice. Downregulating TIMP2 could antagonize these effects and attenuate kidney injury. We also found that the effects of TIMP2 were mediated through the NF-κB pathway. TIMP2 is reported to be abundantly expressed in multiple human tissues such as the ovary, endometrium, gall bladder, and urinary bladder. In addition, TIMP2 acts on multiple genes, including matrix metalloproteinase (MMP), mitogen-activated protein kinase (MAPK), and β-catenin melanoma, and plays different roles in various organs, such as growth-stimulatory activity, revitalizing hippocampal function in aged mice, and promoting leukemia cell invasion [18,[22], [23], [24]]. Thus far, the knowledge of TIMP2 in the kidney has been limited to urinary biomarkers to predict AKI, and its exact role in kidney injury remains unknown [25]. In the present study, we used LPS-stimulated HK-2 Benazepril and CLP-induced AKI mice and reported a prominent upregulation of TIMP2 expression primarily in renal tubular cells. This CLP model is well known to induce septic AKI [26,27]. We confirmed that TIMP2 expression increases during the early stages of AKI and shows a significant positive correlation with the severity of renal dysfunction in septic mice. The development of AKI is related to the mechanisms of inflammation, oxidative stress, and apoptosis in cellular and molecular pathways [6,28,29]. In recent years, there has been increasing recognition that systemic inflammation can contribute to the induction of AKI [30]. Clinical studies indicate that plasma and urinary cytokine levels are correlated with degrees of tubular dysfunction, the risk of developing AKI, and subsequent patient outcomes [31,32]. In the present study, both mRNA and protein levels of IL-1β, IL-6, and TNF-α were significantly increased in the LPS-stimulated HK-2 cells and in mouse kidney tissue after CLP. Previous studies have also reported that the mRNA transcription levels of IL-1, IL-6, and TIMP2 increased significantly in renal tubular epithelial cells in septic rats [33]. Higher levels of IL-6 correlate with mortality and kidney injury [34], and transgenic knockouts of IL-6 ameliorated renal injury [35]. IL-6 deficiency attenuated neutrophil accumulation and caused mice to become relatively resistant to nephrotoxin-induced AKI and ischemic AKI as measured by the serum creatinine level and histological analysis [36]. Elevated renal inflammation was associated with increased expression of TNF-α and TIMP2 in a rat model of chronic kidney disease [37]. In our study, TIMP2 expression was increased in the presence of cytokines such as IL-6, IL-1β, TNF-α, and IFN-γ. Our results are consistent with the studies of Lizárraga et al., which showed that the activation of Benazepril NF-κB was regulated by TIMP2 in A549 lung epithelial cells [38]. In melanoma cells, overexpression of TIMP2 could upregulate NF-κB activity [39]. In our study, the activation of the NF-κB pathway induced by LPS stimulation was partially decreased by silencing TIMP2 in HK-2 cells. We also found that in response to LPS stimulation, tubular epithelial apoptosis was induced; this finding was also reported by other researchers [40]. TIMP2 silencing resulted in decreased LPS-induced apoptosis, suggesting a pro-apoptotic role of TIMP2, perhaps through promoting cytokine synthesis.