Auto- and paracrine rewiring of NIX-mediated mitophagy by insulin-like growth factor-binding protein 7 in septic AKI escalates inflammation-coupling tubular damage.

AIMS: Inflammation-coupling tubular damage (ICTD) contributes to pathogenesis of septic acute kidney injury (AKI), in which insulin-like growth factor-binding protein 7 (IGFBP-7) serves as a biomarker for risk stratification. The current study aims to discern how IGFBP-7 signalling influences ICTD, the mechanisms that underlie this process and whether blockade of the IGFBP-7-dependent ICTD might have therapeutic value for septic AKI. MATERIALS AND METHODS: In vivo characterization was carried out in B6/JGpt-Igfbp7em1Cd1165/Gpt mice subjected to cecal ligation and puncture (CLP). Transmission electron microscopy, immunofluorescence, flow cytometry, immunoblotting, ELISA, RT-qPCR and dual-luciferase reporter assays were used to determine mitochondrial functions, cell apoptosis, cytokine secretion and gene transcription. KEY FINDINGS: ICTD augments the transcriptional activity and protein secretion of tubular IGFBP-7, which enables an auto- and paracrine signalling via deactivation of IGF-1 receptor (IGF-1R). Genetic knockout (KO) of IGFBP-7 provides renal protection, improves survival and resolves inflammation in murine models of cecal ligation and puncture (CLP), while administering recombinant IGFBP-7 aggravates ICTD and inflammatory invasion. IGFBP-7 perpetuates ICTD in a NIX/BNIP3-indispensable fashion through dampening mitophagy that restricts redox robustness and preserves mitochondrial clearance programs. Adeno-associated viral vector 9 (AAV9)-NIX short hairpin RNA (shRNA) delivery ameliorates the anti-septic AKI phenotypes of IGFBP-7 KO. Activation of BNIP3-mediated mitophagy by mitochonic acid-5 (MA-5) effectively attenuates the IGFBP-7-dependent ICTD and septic AKI in CLP mice. SIGNIFICANCE: Our findings identify IGFBP-7 is an auto- and paracrine manipulator of NIX-mediated mitophagy for ICTD escalation and propose that targeting the IGFBP-7-dependent ICTD represents a novel therapeutic strategy against septic AKI.

Caspase-1-Inhibitor AC-YVAD-CMK Inhibits Pyroptosis and Ameliorates Acute Kidney Injury in a Model of Sepsis.

OBJECTIVE: To observe the protective effect of AC-YVAD-CMK on sepsis-induced acute kidney injury in mice and to explore its possible mechanisms primarily. METHODS: Eighteen male C57BL/6 mice were randomly divided into sham-operated group (Control), cecal ligation and puncture group (CLP), and CLP model treated with AC-YVAD-CMK group (AC-YVAD-CMK) (n = 6 in each group). Mice were sacrificed at 24 h after operation, and blood and kidney tissue samples were collected for analyses. Histologic changes were determined microscopically following HE staining. The expression of Ly-6B and CD68 was investigated using immunohistochemistry. Serum concentrations of creatinine (sCR) and blood urea nitrogen (BUN) were measured. Serum levels of interleukin-1ß (IL-1ß), interleukin-18 (IL-18), TNF-α, and interleukin-6 (IL-6) were determined by ELISA. The expressions of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues were investigated using Western blot. Immunofluorescence staining was used to detect the expression of GSDMD protein in renal tissues. RESULTS: AC-YVAD-CMK treatment significantly alleviates sepsis-induced acute kidney injury, with decreased histological injury in renal tissues, suppresses the accumulation of neutrophils and macrophages in renal tissues, and decreased sCR and BUN level (P < 0.05). Attenuation of sepsis-induced acute kidney injury was due to the prohibited production of inflammatory cytokines and decrease expression of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues. In addition, AC-YVAD-CMK treatment significantly reduced the expression of GSDMD in renal tissues compared to those observed in controls (P < 0.05). CONCLUSIONS: We demonstrated a marked renoprotective effect of caspase-1-inhibitor AC-YVAD-CMK in a rat model of sepsis by inhibition of pyroptosis.

miR-34b-3p protects against acute kidney injury in sepsis mice via targeting ubiquitin-like protein 4A.

MicroRNAs (miRNAs) have been reported as a diagnostic markers for sepsis, and miRNAs have also been found to play a regulatory role in sepsis-induced acute kidney injury (AKI). However, the regulatory effect and mechanism of miR-34b-3p on AKI remains elusive. First, sepsis mice with AKI was established via cecal ligation puncture (CLP), and verified through hematoxylin-eosin staining, determination of tumor necrosis factor-α (TNF-α), interleukin (IL)-6/1ß and serum levels of alanine aminotransferase (ALT) and blood urea nitrogen (BUN). Data showed that CLP-induced mice demonstrated increased ALT, BUN, TNF-α, IL-1ß, and IL-6 with injured pathological morphology of kidney tissues. Second, lipopolysaccharide (LPS) treatment elevated TNF-α, IL-1ß, and IL-6 contents in rat mesangial cells (RMCs). MiR-34b-3p was downregulated in both CLP-induced mice and LPS-induced RMCs. Third, target gene of miR-34b-3p was verified as ubiquitin-like protein 4A (UBL4A), and UBL4A was upregulated in LPS-induced RMCs. MiR-34b-3p could inhibit UBL4A expression and decreased TNF-α, IL-1ß and IL-6 contents in LPS-induced RMCs, while overexpression of UBL4A counteract with the suppressive effects of miR-34b-3p on the protein expression. Moreover, transcriptional activity of UBL4A-induced NF-κB was decreased by miR-34b-3p. Lastly, in vivo injection of miR-34b-3p agomir improved CLP-induced kidney tissues injury with declined ALT, BUN, TNF-α, IL-1ß, IL-6, and UBL4A. In general, miR-34b-3p overexpression could alleviate AKI in sepsis mice through downregulation of UBL4A/NF-κB, providing potential therapeutic strategy for AKI.

Regulatory mechanism of NOV/CCN3 in the inflammation and apoptosis of lung epithelial alveolar cells upon lipopolysaccharide stimulation.

Lipopolysaccharide (LPS) induces stress inflammation and apoptosis. Pulmonary epithelial cell apoptosis, which accelerates the progression of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), is the leading cause of mortality in patients with ALI/ARDS. The nephroblastoma overexpressed protein (CCN3), an inflammatory modulator, is reported to be a biomarker in ALI. Using the LPS-induced ALI model, this study investigated the expression of CCN3 and its possible molecular mechanism in lung alveolar epithelial cell inflammation and apoptosis. Our data revealed that LPS treatment greatly increased the level of CCN3 in A549 cells. The A549 cells were transfected with specific CCN3 small interfering RNA (siRNA) using transfection reagent. CCN3 siRNA not only largely attenuated the expressions of the inflammatory cytokines interleukin (IL)-1ß and transforming growth factor (TGF)-ß1, but also reduced the apoptotic rate of the AEC II cells and affected the expressions of the apoptosis-associated proteins (Bcl-2 and caspase-3). Furthermore, CCN3 knockdown greatly inhibited the activation of nuclear factor-κB p65 in A549 cells. In addition, TGF-ß/p-Smad inhibitor (TP0427736) and NF-κB inhibitor (PDTC) significantly attenuated the expression level of CCN3 in A549 cells. In conclusion, our data indicated that CCN3 siRNA affected downstream signal through TGF-ß/ p-Smad or NF-κB pathway, leading to the inhibition of cell inflammation and apoptosis in human alveolar epithelial cells.

Role of Sirolimus in renal tubular apoptosis in response to unilateral ureteral obstruction.

Renal tubule cell apoptosis plays a pivotal role in the progression of chronic renal diseases. The previous study indicates that Sirolimus is effective on unilateral ureteral obstruction (UUO)-induced renal fibrosis. However, the role of Sirolimus in renal tubular apoptosis induced by UUO has not yet been addressed. The aim of this study was to determine the role of Sirolimus in renal tubular apoptosis induced by UUO. Male Sprague-Dawley rats were divided into three groups, sham-operated rats, and after which unilateral ureteral obstruction (UUO) was performed: non-treated and sirolimus-treated (1mg/kg). After 4, 7 and 14 d, animals were sacrificed and blood, kidney tissue samples were collected for analyses. Histologic changes and interstitial collagen were determined microscopically following HE and Masson's trichrome staining. The expression of PCNA was investigated using immunohistochemistry and the expression of Bcl-2, Bax, caspase-9, and caspase-3 were investigated using Western blot in each group. Tubular apoptotic cell deaths were assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Sirolimus administration resulted in a significant reduction in tubulointerstitial fibrosis scores. After UUO, there was an increase in tubular and interstitial apoptosis in untreated controls as compared to Sirolimus treatment rats (P<0.05). In addition, the expression of PCNA, Bcl-2, Bax, caspase-9, and caspase-3 in obstructed kidney was characterized by immunohistochemistry and Western blot analyses demonstrating that sirolimus treatment significantly reduced PCNA, Bax, caspase-9 and cleaved caspase-3 expression compared to those observed in controls (P<0.05), whereas, Bcl-2 in the obstructed kidney were decreased in untreated controls compared to Sirolimus treatment rats subjected to the same time course of obstruction (P<0.05). We demonstrated a marked renoprotective effect of sirolimus by inhibition of UUO-induced renal tubular apoptosis in vivo.