Kidney Injury In Septic Mice Research Articles

Shenfu injection ameliorates endotoxemia-associated endothelial dysfunction and organ injury via inhibiting PI3K/Akt-mediated glycolysis

Ethnopharmacological relevanceMicrocirculatory dysfunction is one of the main characteristics of sepsis. Shenfu Injection (SFI) as a traditional Chinese medicine is widely applied in clinical severe conditions. Recent studies have shown that SFI has the ability to ameliorate sepsis-induced inflammation and to improve microcirculation perfusion. Aim of the studyThis study aims to investigate the underlying mechanism of SFI for ameliorating sepsis-associated endothelial dysfunction and organ injury. Materials and methodsSide-stream dark-field (SDF) imaging was used to monitor the sublingual microcirculation of septic patients treated with or without SFI. Septic mouse model was used to evaluate the effects of SFI in vivo. Metabolomics and transcriptomics were performed on endothelial cells to identify the underlying mechanism for SFI-related protective effect on endothelial cells. ResultsSFI effectively abolished the disturbance and loss of sublingual microcirculation in septic patients. Twenty septic shock patients with or without SFI administration were enrolled and the data showed that SFI significantly improved the levels of total vessel density (TVD), perfused vessel density (PVD), microvascular flow index (MFI), and the proportion of perfused vessels (PPV). The administration of SFI significantly decreased the elevated plasma levels of Angiopoietin-2 (Ang2) and Syndecan-1, which are biomarkers indicative of endothelial damage in sepsis patients. In the mouse septic model in vivo, SFI inhibited the upregulation of endothelial adhesion molecules and Ly6G + neutrophil infiltration while restored the expression of VE-Cadherin in the vasculature of the lung, kidney, and liver tissue. Additionally, SFI reduced the plasma levels of Ang2, Monocyte Chemoattractant Protein-1(MCP1), and Interleukin-6 (IL6), and alleviated liver and kidney injury in septic mice. Moreover, SFI significantly inhibited the inflammatory activation and increased permeability of endothelial cells induced by endotoxins in vitro. By performing metabolomics and transcriptomics, we identified the activation of PI3K/Akt-mediated glycolysis as the underlying mechanism for SFI-related protective effect on endothelial cells. ConclusionsOur findings revealed that SFI may improve microcirculation perfusion and endothelial function in sepsis via inhibiting PI3K/Akt-mediated glycolysis, providing theoretical evidence for the clinical application of SFI.

Chlorogenic acid alleviates acute kidney injury in septic mice by inhibiting NLRP3 inflammasomes and the caspase-1 canonical pyroptosis pathway

To investigate the role of caspase-1-medicated canonical pyroptosis pathway in chlorogenic acid (CGA) treatment of acute kidney injury (AKI) in mice. Twenty-four C57Bl/6J mice were randomized into sham-operated group, cecal ligation and puncture (CLP) group, CLP+dexamethasone group (CLP+DXM group), and CLP+CGA group (n=6) and subjected to either sham operation (laparotomy only) or CLP. After modeling the mice received intravenous infusion of 10 mg/kg normal saline (in sham and CLP groups), 1 μg/kg dexamethasone or 15 mg/kg of chlorogenic acid for 6 h delivered using an intravenous pump. Eight hours after the infusion, renal morphology and histology, renal cell apoptosis, and the renal function parameters such as urea nitrogen (BUN), creatinine (Scr), and kidney injury molecule 1 (KIM-1) were compared among the 4 groups; the 7-day survival rates of the mice were recorded, and the expressions of NLRP3 inflammasomes and key proteins of the caspase-1 pathway in the renal tissue were detected. CGA treatment significantly improved the 7-day survival rate, reduced renal pathologies of the septic mice (P < 0.05), and lowered the levels of BUN, Scr, KIM-1, NLRP3 inflammasome and expressions of key proteins of the caspase-1 pathway. CGA alleviates AKI in mice with CLP-induced sepsis by inhibiting NLRP3 inflammasomes and the caspase-1 signaling pathway.

Maresin-1 Attenuates Sepsis-Associated Acute Kidney Injury via Suppressing Inflammation, Endoplasmic Reticulum Stress and Pyroptosis by Activating the AMPK/SIRT3 Pathway.

Sepsis-associated acute kidney injury (SA-AKI) is a common complication in patients with sepsis, triggering high morbidity and mortality. Maresin-1 (MaR1) is a pro-resolution lipid mediator that promotes the resolution of acute inflammation and protects organs from inflammation. In this study, we established an SA-AKI model using cecal ligation and puncture (CLP) and investigated the effect and mechanism of MaR1. The blood and kidneys were harvested 24 hours after surgery. The blood biochemical/routine indicators, renal function, SA-AKI-related pathophysiological processes, and AMPK/SIRT3 signaling in septic mice were observed by histological staining, immunohistochemical staining, Western blot, qPCR, ELISA and TUNEL Assay. MaR1 treatment alleviated kidney injury in septic mice, reflected in improved pathological changes in renal structure and renal function. MaR1 treatment decreased the levels of serum creatinine (sCr) and blood urea nitrogen (BUN) and the expressions of KIM-1, NGAL and TIMP-2, which were related to kidney injury, while inhibited the expressions of inflammatory factors TNF-α, IL-1β and IL-6. The expression of endoplasmic reticulum stress-related indicators p-PERK/PERK, GRP78, p-EIF2α/EIF2α, ATF4, CHOP, and pyroptosis-related indicators Caspase-1, NLRP3, GSDMD, IL-18, and IL-1β also decreased after MaR1 treatment. The mechanism may be related to the activation of the AMPK/SIRT3 signaling pathway, and an AMPK inhibitor (compound C) partially reverses MaR1's protective effects in septic mice. Taken together, these findings suggest that MaR1 may partially ameliorate SA-AKI by activating the AMPK/SIRT3 signaling pathway, providing a potential new perspective for research on SA-AKI.

Protective effect and mechanism of Baicalin on acute kidney injury in septic mice

To observe the protective effect and mechanism of different doses of Baicalin (BAI) on acute kidney injury (AKI) in septic mice. According to the random number table, 100 mice were divided into sham operation group (Sham group), cecal ligation and perforation (CLP) induced sepsis model group (CLP group) and BAI pretreatment groups. The mice in BAI pretreatment groups were divided into low-, medium- and high-dose groups (BAI-L+CLP, BAI-M+CLP, BAI-H+CLP groups), with 20 mice in each group. A murine sepsis associated-acute kidney injury (SA-AKI) model was reproduced using CLP. The mice in the Sham group were only opened and closed the abdomen, without ligating or perforating the cecum. The mice in the BAI pretreatment groups were given BAI 25, 50 and 100 mg/kg daily for 3 days, and CLP was performed at 6 hours after administration of BAI at the 3rd day to reproduce sepsis model. The mice in the Sham group and CLP group were given the same amount of distilled water as control. Ten mice were sacrificed at 24 hours after operation to collect orbital blood for renal function determination [serum creatinine (SCr), blood urea nitrogen (BUN), plasma neutrophil gelatinase-associated lipocalin (pNGAL) and plasma kidney injury molecule-1 (pKIM-1)] by enzyme linked immunosorbent assay (ELISA). The kidney tissue was collected to observe the kidney tissue injury under light microscope after hematoxylin-eosin (HE) staining. The TdT-mediated dUTP nick-end labeling (TUNEL) was used to detect the apoptosis of renal tubular epithelial cells. Western blotting was used to detect the expression of cell FLICE like inhibitory protein (c-FLIP) in renal tissue. The remaining 10 mice in each group were used to calculate the survival rate of 7 days after operation. The renal tubular epithelial cells in the CLP group were massively degenerated with necrosis, the renal tubular lumen was significantly expanded, and inflammatory cells were widely infiltrated in the renal interstitium. Furthermore, the renal function deteriorated rapidly. Compared with the CLP group, the renal function of mice pretreated with low dose of BAI was improved, but the difference was not significant. Compared with the CLP group, the renal function in the mice pretreated with medium and high doses of BAI was significantly improved, the SCr, BUN, pNGAL and pKIM-1 were significantly reduced [SCr (μmol/L): 135.16±5.18, 125.70±5.26 vs. 170.42±5.42; BUN (mmol/L): 33.59±1.77, 27.29±1.61 vs. 45.68±1.39; pNGAL (μg/L): 91.29±4.68, 73.40±3.77 vs. 131.50±6.55; pKIM-1 (μg/L): 6.34±0.30, 5.51±0.35 vs. 8.03±0.29; all P < 0.01], the pathological injury of renal tissue was significantly decreased, the apoptotic number of renal tubular epithelial cells was significantly reduced (cells/HP: 16.20±0.49, 13.10±0.66 vs. 29.60±0.49, both P < 0.01), and the expression of c-FLIP protein in renal tissue was significantly increased [c-FLIP protein (c-FLIP/GAPDH): 0.35±0.02, 0.46±0.02 vs. 0.21±0.01, both P < 0.01]. No mouse in the Sham group died within 7 days. Compared with the CLP group, the average survival time of the mice within 7 days in the BAI-L+CLP, BAI-M+CLP and BAI-H+CLP groups was significantly prolonged with a dose-dependent manner (days: 3.5±2.5, 5.4±2.2, 5.9±1.9 vs. 2.1±1.2; Log-Rank test: χ2 = 73.410, P < 0.001). Pretreatment with medium and high doses of BAI can significantly improve the renal function in mice with SA-AKI, decrease the pathological damage and increase the survival of mice, and its mechanism may be related to promoting the increase of c-FLIP protein expression and inhibiting cell apoptosis.

The role of lipoprotein-associated phospholipase A2 in acute kidney injury of septic mice.

BackgroundThis experiment aimed to investigate the role and mechanism of lipoprotein-associated phospholipase A2 (Lp-PLA2) in kidney injury in septic mice induced by cecal ligation and perforation (CLP).MethodsMale BALB/c mice were randomly divided into two groups: sham-operation group (Sham group) and septic group (CLP group). The septic model was simulated by cecal ligation and puncture method, but only cecal ligation was used for the sham operation group. The whole serum and renal tissue samples of the mice were collected 24 hours after modeling in both groups. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissue, the renal injury score was recorded, and the creatinine (Cr) and blood urea nitrogen (BUN) levels were detected by automatic biochemical analyzer, while the serum Lp-PLA2 level was detected by enzyme-linked immunosorbent assay (ELISA). The 7-day survival rate and the survival curve of the two groups were statistically analyzed.ResultsCompared with the Sham group, the pathological score of renal injury in the CLP Group was higher, the level of Lp-PLA2 in serum was significantly increased (all P<0.01), and the expression of Lp-PLA2 in renal tissue was significantly elevated (all P<0.01). Furthermore, the 7-day survival rate of the Sham group was 90%, while that of CLP group was 25%.ConclusionsThe expression level of Lp-PLA2 in blood and kidney tissue of septic mice was increased and correlated with prognosis. However, the predictive value of Lp-PLA2 for prognosis in septic mice needs further study.

Protective effects of Klotho protein on acute kidney injury in septic mice and its mechanism

To investigate the protective effects of Klotho protein, a kind of single-pass transmembrane protein, on acute kidney injury (AKI) in septic mice and its mechanism. Sixty SPF healthy male C57BL/6 mice (6-8 weeks) were randomly divided into sham operation group (Sham group), sepsis model group (CLP group) and Klotho protein injection group (CLP+KL group), with 20 in each group. The septic AKI mice model was established by cecal ligation and puncture (CLP); Sham group had the same procedure except that the cecal was not ligated. The CLP+KL group was received Klotho protein (0.02 mg/kg) by intraperitoneal consecutive injection for 4 days after operation; Sham group and CLP group were injected with the same amount of saline. Blood samples were obtained at 24 hours after operation, the levels of serum creatinine (SCr) and urea nitrogen (BUN) were measured by sarcosine oxidase and urease method. The mice were sacrificed under anesthesia at 5 days after operation to harvest renal tissues, and the pathological damage of the kidney was evaluated by hematoxylin-eosin (HE) staining. The ultrastructure of mitochondria in mouse renal tubular epithelial cells was observed under transmission electron microscope. The levels of reduced glutathione hormone (GSH), malondialdehyde (MDA) and nitric oxide synthase (NOS) in mitochondrion were determined by micro-enzyme method, thiobarbituric acid method, colorimetry method, respectively. The protein expressions of Klotho, Bcl-2 and cytochrome C (Cyt C) were detected by Western Blot. The pathological structure of the kidneys in the Sham group was clear and intact. Compared with the Sham group, the renal tissue edema of the mice in the CLP group was significant, and the transparent tube type was observed in the small lumen, and the interstitial inflammatory cells infiltrated; the levels of SCr and BUN were significantly increased [SCr (μmol/L): 182.60±6.97 vs. 47.20±5.37, BUN (mmol/L): 53.70±5.12 vs. 18.70±2.62, both P < 0.01]; the mitochondria were swollen and deformed, the sputum structure was destroyed, the matrix density was decreased, the outer membrane was lost, and the levels of MDA, GSH and NOS were significantly increased [MDA (μmol/g): 1.172±0.046 vs. 0.746±0.094, GSH (μmol/g): 5.765±0.059 vs. 4.223±0.072, NOS (kU/g): 0.91±0.05 vs. 0.68±0.03, all P < 0.01]; the protein expressions of Klotho and Bcl-2 in renal tissue were decreased, and the protein expression of Cyt C was increased (Klotho/β-actin: 0.188±0.020 vs. 0.538±0.024, Bcl-2/β-actin: 0.311±0.010 vs. 0.391±0.015, Cyt C/β-actin: 0.226±0.010 vs. 0.135±0.006, all P < 0.01). Comparing with the CLP group, the glomerular and tubular tissue epithelial edema and the small lumen in the CLP+KL group were reduced; the levels of SCr and BUN were significantly decreased [SCr (μmol/L): 85.70±7.23 vs. 182.60±6.97, BUN (mmol/L): 35.30±3.50 vs. 53.70±5.12, both P < 0.01]; the mitochondrial structure was relatively intact; the levels of MDA, GSH and NOS were significantly decreased [MDA (μmol/g): 0.958±0.072 vs. 1.172±0.046, GSH (μmol/g): 4.756±0.107 vs. 5.765±0.059, NOS (kU/g): 0.79±0.02 vs. 0.91±0.05, all P < 0.01]; the protein expressions of Klotho, Bcl-2 were significantly increased, but the protein expression of Cyt C was significantly decreased (Klotho/β-actin: 0.336±0.011 vs. 0.188±0.020, Bcl-2/β-actin: 0.474±0.017 vs. 0.311±0.010, Cyt C/β-actin: 0.168±0.006 vs. 0.226±0.010, all P < 0.01). Klotho protein has significant protective effects on AKI in septic mice, and its mechanism is related to maintaining mitochondrial structural integrity and oxidative stress response.

Pterostilbene attenuates acute kidney injury in septic mice.

Acute kidney injury (AKI) is a severe complication of sepsis with a high mortality and morbidity. Pterostilbene (Pte) has been suggested to confer anti-apoptotic and anti-inflammatory effects. In the current study, the effects of Pte on AKI were evaluated in septic mice. Cecal ligation and puncture surgery was performed to induce sepsis. The results suggested that Pte administration significantly decreased the levels of serum urea nitrogen and creatinine, and improved the survival rate of septic mice. Additionally, the renal injury induced by sepsis was attenuated by pterostilbene treatment. Notably, pterostilbene reduced Bcl-2-associated X protein expression, and levels of interleukin-1β and tumor necrosis factor-α, and upregulated B-cell lymphoma 2 expression. The results indicate that pterostilbene may serve as a potential therapeutic candidate for the treatment of AKI induced by sepsis.

Anticancer Agent 2-Methoxyestradiol Improves Survival in Septic Mice by Reducing the Production of Cytokines and Nitric Oxide

Cytokine production is critical in sepsis. 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol, inhibits hypoxia-inducible factor 1α (HIF-1α) and is an antiangiogenic and antitumor agent. We investigated the effect of 2ME2 on cytokine production and survival in septic mice. Using i.p. LPS or cecal ligation and puncture (CLP), sepsis was induced in BALB/c mice that were simultaneously or later treated with 2ME2 or vehicle. Twelve hours after the LPS injection, serum and peritoneal fluid cytokine and nitric oxide (NO) levels were analyzed using enzyme-linked immunosorbent assay and the Griess reaction. Lung injuries were histologically analyzed, and liver and kidney injuries were biochemically analyzed. Survival was determined 7 days after LPS injection or CLP procedure. In vivo and in vitro effects of 2ME2 on LPS-induced macrophage inflammation were determined. The effect of 2ME2 on HIF-1α expression, nuclear factor κB (NF-κB), and inducible NO synthase (iNOS) in LPS-treated RAW264.7 cells, a murine macrophage cell line, was determined using Western blotting. 2-Methoxyestradiol treatment reduced LPS-induced lung, liver, and kidney injury. Both early and late 2ME2 treatment prolonged survival in LPS- and CLP-induced sepsis. 2-Methoxyestradiol significantly reduced IL-1β, IL-6, TNF-α, and NO levels in septic mice as well as in LPS-stimulated peritoneal macrophages. 2-Methoxyestradiol treatment also reduced the LPS-induced expression of HIF-1α, iNOS, and pNF-κB in RAW264.7 cells, as well as iNOS and pNF-κB expression in siHIF-1α-RAW264.7 cells. 2-Methoxyestradiol prolongs survival and reduces lung, liver, and kidney injury in septic mice by inhibiting iNOS/NO and cytokines through HIF-1α and NF-κB signaling.