Mouse Model Of Acute Kidney Injury Research Articles

RIPK3 inhibitor-AZD5423 alleviates acute kidney injury by inhibiting necroptosis and inflammation

Acute kidney injury (AKI) is a clinical syndrome that is defined as a sudden decline in renal function and characterized by inflammation and programmed cell death of renal tubular epithelial cells. Necroptosis is a form of regulated cell death that requires activation of receptor interacting protein kinase 3 (RIPK3) and its phosphorylation of the substrate MLKL. RIPK3 plays an important role in acute kidney injury, and hence developing its inhibitors is considered as one of the promising strategies aimed at prevention and treatment of AKI. Recently, we discovered AZD5423 as a novel potent RIPK3 inhibitor using a computer-aided hybrid virtual screening strategy according to three-dimensional structure of RIPK3. Our findings revealed that AZD5423 strongly inhibits activation of RIPK3, and MLKL phosphorylation upon cisplatin-, hypoxia/reoxygenation (H/R)- and TNF-α stimuli as compared with GSK872, which is a previously identified RIPK3 inhibitor. Importantly, AZD5423 exerts effective protection against cisplatin- and ischemia/reperfusion (I/R)-induced AKI mouse model. The results of cellular thermal shift assay and experiments in RIPK3 knockout cells indicated that AZD5423 could directly target RIPK3 to inhibit RIPK3 kinase activity. Mechanistically, the docking of AZD5423 and RIPK3 suggested that the kinase domain of RIPK3 for Lys50, Arg313, Lys29, Arg37 might form hydrogen bonds with AZD5423. Site-directed mutagenesis further revealed that AZD5423 reduces injury response via interacting with the key RIPK3 amino acid residues of Lys50 and Arg313. In conclusion, our study has demonstrated that AZD5423 may serve as a potent inhibitor of RIPK3 kinase and a promising clinical candidate for AKI treatment.

Regulator of calcineurin 1 deletion attenuates mitochondrial dysfunction and apoptosis in acute kidney injury through JNK/Mff signaling pathway

Ischemia-reperfusion (I/R) induced acute kidney injury (AKI), characterized by excessive mitochondrial damage and cell apoptosis, remains a clinical challenge. Recent studies suggest that regulator of calcineurin 1 (RCAN1) regulates mitochondrial function in different cell types, but the underlying mechanisms require further investigation. Herein, we aim to explore whether RCAN1 involves in mitochondrial dysfunction in AKI and the exact mechanism. In present study, AKI was induced by I/R and cisplatin in RCAN1flox/flox mice and mice with renal tubular epithelial cells (TECs)-specific deletion of RCAN1. The role of RCAN1 in hypoxia-reoxygenation (HR) and cisplatin-induced injury in human renal proximal tubule epithelial cell line HK-2 was also examined by overexpression and knockdown of RCAN1. Mitochondrial function was assessed by transmission electron microscopy, JC-1 staining, MitoSOX staining, ATP production, mitochondrial fission and mitophagy. Apoptosis was detected by TUNEL assay, Annexin V-FITC staining and Western blotting analysis of apoptosis-related proteins. It was found that protein expression of RCAN1 was markedly upregulated in I/R- or cisplatin-induced AKI mouse models, as well as in HR models in HK-2 cells. RCAN1 deficiency significantly reduced kidney damage, mitochondrial dysfunction, and cell apoptosis, whereas RCAN1 overexpression led to the opposite phenotypes. Our in-depth mechanistic exploration demonstrated that RCAN1 increases the phosphorylation of mitochondrial fission factor (Mff) by binding to downstream c-Jun N-terminal kinase (JNK), then promotes dynamin related protein 1 (Drp1) migration to mitochondria, ultimately leads to excessive mitochondrial fission of renal TECs. In conclusion, our study suggests that RCAN1 could induce mitochondrial dysfunction and apoptosis by activating the downstream JNK/Mff signaling pathway. RCAN1 may be a potential therapeutic target for conferring protection against I/R- or cisplatin-AKI.

A novel contrast-induced acute kidney injury mouse model based on low-osmolar contrast medium

The contrast-induced acute kidney injury (CI-AKI) has been becoming the third common cause of hospital-acquired acute kidney injury. An ideal animal model is essential for understanding the pathophysiology of CI-AKI. Previous CI-AKI studies were mostly performed on rats with high-osmolar contrast medium (HOCM), which is unsuitable for transgenic researches. This study provides a novel, efficient and reproducible CI-AKI model which was developed in mouse by administrating a low-osmolar contrast medium (LOCM). First of all, we applied the frequently used pretreatments (uninephrectomy and water deprivation), which combined with HOCM on rats could induce CI-AKI, on mice with LOCM. Secondly, we attempted to find a novel pretreatment suitable for mouse and LOCM by combining two classic pretreatments(uninephrectomy, water deprivation and furosemide administration). Finally, we evaluate the kidney damage of the novel model. We found that this mouse model possessed a significant reduction in renal function, severe renal tissue damage, and increased renal tubular cells apoptosis, indicating that LOCM is a feasible inducer for CI-AKI mice model. Taken together, we found that uninephrectomy (UPHT) combined with 24 h water deprivation and furosemide administration 20 min before LOCM (iohexol, 10 ml/kg) application is a feasible pretreatment to establish a novel CI-AKI mouse model.

Exosomal lncRNA TUG1 derived from human urine-derived stem cells attenuates renal ischemia/reperfusion injury by interacting with SRSF1 to regulate ASCL4-mediated ferroptosis

BackgroundHuman urine-derived stem cells (USCs)-derived exosomes (USC-Exo) could improve kidney ischemia/reperfusion injury (IRI), while the underlying mechanisms of this protective effect remain unclear.MethodsHuman USCs and USC-Exo were isolated and verified by morphology and specific biomarkers. The effects of USC-Exo on ferroptosis and kidney injury were detected in the IRI-induced acute kidney injury (AKI) model in C57BL/6 mice. The effects of USC-Exo on ferroptosis and lncRNA taurine-upregulated gene 1 (TUG1) were detected in hypoxia/reoxygenation (H/R)-treated human proximal tubular epithelial cells (HK-2). The interaction of SRSF1 and TUG1, ACSL4 was checked via RNA pull-down/RIP and RNA stability assays. The effects of LncRNA TUG1 on SRSF1/ACSL4-mediated ferroptosis were verified in H/R-treated HK-2 cells and the IRI-induced AKI mouse models.ResultsUSC-Exo treatment improved kidney injury and ameliorated ferroptosis in IRI-induced AKI mouse models. USC-Exo were rich in lncRNA TUG1, which suppressed ferroptosis in HK-2 cells exposed to H/R. Mechanistically, lncRNA TUG1 regulates the stability of ACSL4 mRNA by interacting with RNA-binding protein SRSF1. In addition, SRSF1 upregulation or ACSL4 downregulation partially reversed the protective effect of lncRNA TUG1 on ferroptosis in H/R-treated HK-2 cells. Further, ACSL4 upregulation partially reversed TUG1’s repression on kidney injury and ferroptosis in IRI-induced AKI mice.ConclusionCollectively, lncRNA TUG1 carried by USC-Exo regulated ASCL4-mediated ferroptosis by interacting with SRSF1 and then protected IRI-induced AKI. Potentially, USC-Exo rich in lncRNA TUG1 can serve as a promising therapeutic method for IRI-AKI.

Comprehensive Network-Based Analyses Reveal Novel Renal Function-Related Targets in Acute Kidney Injury.

Background: Acute kidney injury (AKI) is a common clinical syndrome with limited methods of treatment and diagnosis. Although several molecules associated with AKI have been discovered, molecular mechanisms underlying AKI still remain unclear. Weighted gene co-expression network analysis (WGCNA) is a novel method to uncover the relationship between co-expression genes and clinical traits at the system level. Methods: First, by employing WGCNA in transcriptional data on 30 patients with well/poor functioning kidney graft, we identified two co-expression modules that were significantly related to serum creatinine (SCr). Second, based on the modules, potential small molecular compound candidates for developing targeted therapeutics were obtained by connectivity map analysis. Furthermore, multiple validations of expression in space/time were carried out with two classical AKI models in vivo and other five databases of over 152 samples. Results: Two of the 14 modules were found to be closely correlated with SCr. Function enrichment analysis illustrated that one module was enriched in the immune system, while the other was in the metabolic process. Six key renal function-related genes (RFRGs) were finally obtained. Such genes performed well in cisplatin-induced or cecal ligation and puncture-induced AKI mouse models. Conclusion: The analysis suggests that WGCNA is a proper method to connect clinical traits with genome data to find novel targets in AKI. The kidney tissue with worse renal function tended to develop a “high immune but low metabolic activity” expression pattern. Also, ACSM2A, GLYAT, CORO1A, DPEP1, ALDH7A1, and EPHX2 are potential targets of molecular diagnosis and treatment in AKI.

Farnesoid X receptor protects against cisplatin-induced acute kidney injury by regulating the transcription of ferroptosis-related genes

The side effects of cisplatin, a widely used chemotherapeutic agent, include nephrotoxicity. Previous studies have reported that cisplatin induces ferroptosis and lipid peroxide accumulation. Ferroptosis, a type of regulated cell death, is characterized by iron-dependent lipid peroxidation. Although previous studies have examined the regulation of ferroptosis in acute kidney injury (AKI), the regulatory mechanism of ferroptosis has not been elucidated. Here, the ability of activated farnesoid X receptor (FXR) to attenuate cisplatin-induced AKI through the regulation of ferroptosis was examined. FXR deficiency exhibited more ferroptosis responses, such as increase in lipid peroxidation, iron content and heme oxygenase 1 protein, and a decrease in glutathione/glutathione disulfide ratio and glutathione peroxidase 4 levels in HK2 cells and mice. Increased blood urea nitrogen, serum creatinine, and ferroptotic responses in the cisplatin-induced AKI mouse model were mitigated upon treatment with the FXR agonist GW4064 but were exacerbated in FXR knockout mice. RNA sequencing analysis revealed that ferroptosis-associated genes were novel targets of FXR. FXR agonist upregulated the expression of lipid and glutathione metabolism-related genes and downregulated cell death-related genes. Additionally, chromatin immunoprecipitation assays, using mice renal tissues, revealed that agonist-activated FXR could bind to its known target genes (Slc51a, Slc51b, Osgin1, and Mafg) and ferroptosis-related genes (Aifm2, Ggt6, and Gsta4). Furthermore, activated FXR-dependent MAFG, a transcriptional repressor, could bind to Hmox1, Nqo1, and Tf in the renal tissues of FXR agonist-treated mice. These findings indicate that activated FXR regulates the transcription of ferroptosis-related genes and protects against cisplatin-induced AKI.

Discovery of a chalcone derivative as potent necroptosis inhibitor for the treatment of acute kidney injury.

Necroptosis, a form of inflammation-related programmed cell death, is a major mechanism of proximal tubular cell injury in acute kidney injury (AKI). Blockade of necroptosis signalling represents a promising strategy for clinical therapy of AKI. Previously, we identified a small molecular receptor-interacting protein kinases (RIPK)1 inhibitor Cpd-71 with nephroprotective activities. To discover more nephroprotective agents, in this study, 20 chalcone derivatives were synthesized and evaluated for their anti-necroptosis and nephroprotective activities. Among the chalcone derivatives, Cpd-2 exhibited the most potent anti-necroptosis activity (IC50 =1.08 μM) and protective activity (EC50 =1.49 μM) through directly binding to RIPK1 and blocking RIPK1-RIPK3-mixed-lineage kinase domain-like protein (MLKL) signalling pathway. Furthermore, Cpd-2 effectively attenuated cisplatin or hypoxia/reoxygenation (H/R)-induced injury and necroptotic inflammation in renal cell models. Moreover, in cisplatin- or ischemia/reperfusion (I/R) induced AKI mouse model, detection of creatinine and urea nitrogen in blood showed that Cpd-2 improved kidney function. Periodic acid-Schiff (PAS) staining and immunofluorescence analysis indicated that Cpd-2 also reduced pathological damage and inhibited inflammatory development in kidney tissues. In summary, although some chalcone derivatives have been reported to prevent kidney injury previously, our present study not only discovered a promising leading compound Cpd-2, but also provided a novel and successful practice for the development of necroptosis inhibitors from natural products derivatives as AKI therapeutic agents.

NCOA2 coordinates with the transcriptional KAT2B-NF-κB partner to trigger inflammation response in acute kidney injury

Dysregulation of multiple genes is an important risk factor for acute kidney injury (AKI). Numerous genes, such as proinflammatory cytokines, intracellular cell adhesion molecules (ICAMs), and nitric oxide synthases (NOSs), are implicated in AKI pathogenesis. However, the molecular mechanisms involved in the dysregulation of these genes are still obscure. Herein, we discovered that two subunits of NF-κB (p50 and p65) couple with lysine acetyltransferase 2B (KAT2B) and nuclear receptor coactivator 2 (NOCA2) to assemble a transcriptional complex in a LPS-induced mouse model of AKI. The NCOA2-KAT2B-NF-κB complex bound to the promoters of some NF-κB target genes, such as interleukin 1 beta (IL-1B), IL-6, tumor necrosis factor alpha (TNFA), ICAM1, vascular cell adhesion molecule 1 (VCAM1), cluster of differentiation 38 (CD38), CD40, CD80, and NOS2, and transactivated their expression. In vitro knockdown of components of the NCOA2-KAT2B-NF-κB complex or blockage of KAT2B by its inhibitors (5-chloro-2-(4-nitrophenyl)-3(2H)-isothiazolone [CNIT] and garcinol) significantly decreased the expression of these NF-κB target genes following LPS treatment. The administration of CNIT and garcinol significantly improved the in vivo outcomes of the AKI mice. Our findings reveal the underlying mechanism of NF-κB target upregulation in the pathogenesis of LPS-induced AKI and identify a new therapeutic strategy for AKI that involves targeting the NCOA2-KAT2B-NF-κB complex.

IL-6 accelerates renal fibrosis after acute kidney injury via DNMT1-dependent FOXO3a methylation and activation of Wnt/β-catenin pathway

Maladaptive repair following acute kidney injury (AKI) can trigger inflammation and fibrosis, while progressive renal interstitial fibrosis is critical for the transition of AKI to chronic kidney disease. The aim of this study was to investigate the effect of pro-inflammatory IL-6 on the renal fibrosis after AKI as well as the underlying mechanism. Bioinformatics analysis was performed to identify potential genes related to renal fibrosis. An in vitro model of AKI was established by hypoxia-reoxygenation (H/R) in mouse renal tubular epithelial cells (mRTECs). Gain- and loss-of-function approaches were employed to identify the effects of IL-6/FOXO3a on the renal fibrosis following AKI. The methylation level of FOXO3a promoter was assessed by methylation specific PCR. An AKI mouse model was established by ischemia-reperfusion. Adenovirus-packaged shRNA targeting IL-6 or FOXO3a was utilized for further exploration of their roles in vivo. IL-6 was highly expressed in H/R-exposed mRTECs and kidney tissues of AKI mice rendered with renal fibrosis. Knockdown of IL-6 slowed the renal fibrosis after AKI. IL-6 induced DNMT1 to promote FOXO3a promoter methylation and thus inhibited FOXO3a expression. This inhibition led to promotion of renal fibrosis through activating the Wnt/β-catenin pathway. Further in vivo results verified that loss of IL-6 disrupted the Wnt/β-catenin pathway through reversing DNMT1-mediated inhibition of FOXO3a and consequently attenuated renal fibrosis. All in all, these results suggest that targeting IL-6 contributes to protection against renal fibrosis after AKI through mediating the DNMT1/FOXO3a/Wnt/β-catenin axis, which may serve as promising target for the treatment of AKI and prevention against chronic kidney disease.

Novel CRBN-Recruiting Proteolysis-Targeting Chimeras as Degraders of Stimulator of Interferon Genes with In Vivo Anti-Inflammatory Efficacy.

The activation of the cyclic GMP-AMP synthase-stimulator of interferon gene (STING) pathway has been associated with the pathogenesis of many autoimmune and inflammatory disorders, and small molecules targeting STING have emerged as a new therapeutic strategy for the treatment of these diseases. While several STING inhibitors have been identified with potent anti-inflammatory effects, we would like to explore STING degraders based on the proteolysis-targeting chimera (PROTAC) technology as an alternative strategy to target the STING pathway. Thus, we designed and synthesized a series of STING protein degraders based on a small-molecule STING inhibitor (C-170) and pomalidomide (a CRBN ligand). These compounds demonstrated moderate STING-degrading activities. Among them, SP23 achieved the highest degradation potency with a DC50 of 3.2 μM. Importantly, SP23 exerted high anti-inflammatory efficacy in a cisplatin-induced acute kidney injury mouse model by modulating the STING signaling pathway. Taken together, SP23 represents the first PROTAC degrader of STING deserving further investigation as a new anti-inflammatory agent.

Inhibition of METTL3 attenuates renal injury and inflammation by alleviating TAB3 m6A modifications via IGF2BP2-dependent mechanisms.

The role of N6-methyladenosine (m6A) modifications in renal diseases is largely unknown. Here, we characterized the role of N6-adenosine-methyltransferase-like 3 (METTL3), whose expression is elevated in renal tubules in different acute kidney injury (AKI) models as well as in human biopsies and cultured tubular epithelial cells (TECs). METTL3 silencing alleviated renal inflammation and programmed cell death in TECs in response to stimulation by tumor necrosis factor-α (TNF-α), cisplatin, and lipopolysaccharide (LPS), whereas METTL3 overexpression had the opposite effects. Conditional knockout of METTL3 from mouse kidneys attenuated cisplatin- and ischemic/reperfusion (I/R)-induced renal dysfunction, injury, and inflammation. Moreover, TAB3 [TGF-β-activated kinase 1 (MAP3K7) binding protein 3] was identified as a target of METTL3 by m6A methylated RNA immunoprecipitation sequencing and RNA sequencing. The stability of TAB3 was increased through binding of IGF2BP2 (insulin-like growth factor 2 binding protein 2) to its m6A-modified stop codon regions. The proinflammatory effects of TAB3 were then explored both in vitro and in vivo. Adeno-associated virus 9 (AAV9)-mediated METTL3 silencing attenuated renal injury and inflammation in cisplatin- and LPS-induced AKI mouse models. We further identified Cpd-564 as a METTL3 inhibitor that had better protective effects against cisplatin- and ischemia/reperfusion-induced renal injury and inflammation than S-adenosyl-l-homocysteine, a previously identified METTL3 inhibitor. Collectively, METTL3 promoted m6A modifications of TAB3 and enhanced its stability via IGF2BP2-dependent mechanisms. Both genetic and pharmacological inhibition of METTL3 attenuated renal injury and inflammation, suggesting that the METTL3/TAB3 axis is a potential target for treatment of AKI.

Hypoxia-inducible factor protects against acute kidney injury via the Wnt/β-catenin signaling pathway.

Promoting adaptive repair in acute kidney injury (AKI) is an effective strategy to prevent the progression from AKI to chronic kidney disease. However, the mechanisms involved in renal repair after AKI remain unclear. In this study, we investigated the role of hypoxia-inducible factor (HIF), an important regulator of ischemic and hypoxic injury, in AKI during the repair phase. We established mouse models of ischemia-reperfusion injury-induced AKI with adaptive repair or maladaptive repair. We found that after injury, activation of HIF in the adaptive repair group was rapid, whereas in the maladaptive repair group HIF activation was relatively delayed, and its expression was significantly lower than that in the adaptive repair group during the early repair phase. To further investigate the mechanism of HIF, we regulated the expression of HIF-1α and HIF-2α in HK-2 cells and EA.hy926 cells, respectively. Silencing HIF expression reduced proliferation and increased apoptosis in cells injured by hypoxia/reoxygenation. Self-healing ability was further reduced due to the downregulation of HIF. Moreover, HIF overexpression had the opposite effect. HIF increased the expression of β-catenin and its downstream target genes. Activation of Wnt/β-catenin by the small-molecule activator SKL2001 mitigated the damaging effect of HIF knockdown, whereas blockade of β-catenin with the inhibitor IWR-1-endo reduced the protective effects of HIF. In conclusion, HIF, which is highly expressed in the early stage after AKI, promotes renal repair by interacting with the Wnt/β-catenin signaling pathway.NEW & NOTEWORTHY We investigated the role of hypoxia-inducible factor (HIF) in acute kidney injury in vivo and in vitro. Expression of HIF in the adaptive repair group was more rapid and sufficient than that in the maladaptive repair group during the early repair phase. HK-2 and EA.hy926 cells treated with hypoxia/reoxygenation were used to elucidate the cross talk between HIF and the Wnt/β-catenin signaling pathway by which HIF played a renoprotective role in acute kidney injury.

Study of Renal Accumulation of Targeted Polycations in Acute Kidney Injury.

Acute kidney injury (AKI) is a global healthcare burden characterized by rapid loss of renal function and high morbidity and mortality. Chemokine receptor CXCR4 participates in the renal infiltration of immune cells following injury and in local inflammatory enhancement. Injured renal tubule cells overexpress CXCR4, which could be used as a target for improved drug delivery in AKI. Plerixafor is a small-molecule CXCR4 antagonist that has shown beneficial effects against AKI and has been previously developed into a polymeric analog (polymeric plerixafor, PP). With the goal of gaining a better understanding of how overall charge and hydrophilicity affect renal accumulation of PP, we have synthesized PP copolymers containing hydroxyl, carboxyl, primary amine, and alkyl moieties using Michael-type addition copolymerization. All synthesized copolymers showed excellent CXCR4-binding and inhibiting ability in vitro and improved cellular uptake in hypoxia-reoxygenation stimulated mouse tubule cells. Analysis of serum protein binding revealed that polymers with hydroxyl group modification showed the least amount of protein binding. Biodistribution of the polymers was tested in a unilateral ischemia reperfusion-induced AKI mouse model. The results showed significant differences in accumulation in the injured kidneys depending on the net charge and hydrophilicity of the polymers. The findings of this study will guide the development of polymeric drug carriers for targeted delivery to injured kidneys for better AKI therapy.

Therapeutic effect of quercetin polymeric nanoparticles on ischemia/reperfusion-induced acute kidney injury in mice

Acute kidney injury (AKI) is known as a sudden episode of kidney injury, which happens suddenly within a few hours or a few days. Quercetin (3,3′,4’,5,7-pentahydroxyflavone) is a flavonoid found in plants. Quercetin is known to have several biological activities, such as anti-oxidant, anti-inflammatory, and anti-carcinogenic effects. However, low water solubility and bioavailability are the limitations of quercetin for its clinical applications. Moreover, ischemia/reperfusion (I/R) injury is a common cause of AKI. There are no satisfactory strategies for I/R-induced AKI. Developing suitable preventive or therapeutic intervention for AKI is an important and urgent issue. We investigated the benefit effect of synthesized polyethylene glycol (PEG) conjugated polyethyleneimine (PEI) nanoparticles for targeted delivery of quercetin on AKI in a mouse model. An I/R-induced AKI mouse model was used to evaluate the therapeutic effect of quercetin polymeric nanoparticles by intravenous injection. Biochemical changes for renal function in blood samples were analyzed. Histological and immunohistochemical changes were also analyzed. The biochemical changes of blood urea nitrogen (BUN), creatinine, and cystatin C were significantly increased in I/R-induced AKI mice, which could be significantly reversed by quercetin polymeric nanoparticles. Quercetin polymeric nanoparticles could also significantly decrease the histological lesions, positive staining for 3-nitrotyrosine and cyclooxygenase-2, and lipid peroxidation in the kidneys of I/R-induced AKI mice. These results demonstrate for the first time that quercetin polymeric nanoparticles possess therapeutic potential for the treatment of I/R-induced AKI in vivo.

MicroRNA-486-5p is implicated in the cisplatin-induced apoptosis and acute inflammation response of renal tubular epithelial cells by targeting HAT1.

The proinflammatory property of cisplatin is potentially destructive andcontributes to the pathogenesis of acute kidney injury (AKI). The role and upstream regulatory mechanism of histone acetyltransferase 1 (HAT1) in acute kidney inflammation are still unknown. We performed RNA sequencing to filter differentially expressed microRNAs (miRNAs) in the kidney tissue of mice with AKI induced by cisplatin and ischemia-reperfusion. Here, we found that miR-486-5p was upregulated and that the expression of HAT1 was reduced in AKI mouse models and injured human renal proximal tubular epithelial cell (HK-2) model induced by cisplatin. miR-486-5p is implicated in cisplatin-induced kidney damage in vivo. Bioinformatics analysis predicted a potential binding site between miR-486-5p and HAT1. The Luciferase reporter assay and Western blot confirmed that miR-486-5p directly targeted the 3'-untranslated region of HAT1 mRNA and inhibited its expression in the cytoplasm of HK-2 cells. In the in vitro study, inhibiting miR-486-5p reduced apoptosis, and the expression of proinflammatory mediators was induced by cisplatin in HK-2 cells. Simultaneously, the downregulation of miR-486-5p inhibited the activation of the toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB). We further found that HAT1 could inhibit apoptosis and the activation of cisplatin on the TLR4/NF-κB pathway and that the upregulation of miR-486-5p reversed this effect. Therefore, the upregulation of miR-486-5p targeting HAT1 promoted the cisplatin-induced apoptosis and acute inflammation response of renal tubular epithelial cells by activating the TLR4/NF-κB pathway, providing a new basis to highlight the potential intervention of regulating the miR-486-5p/HAT1 axis.

An Optimized Two-Herb Chinese Food as Medicine Formula Reduces Cisplatin-Induced Nephrotoxicity in the Treatment of Lung Cancer in Mice.

Chemotherapy is considered a most effective way to treat cancer. However, it is very common that chemotherapy causes unbearable mental and physical side effects to cancer patients, which ultimately reduces the patients’ confidence of overcoming diseases and compromises the treatment of chemotherapy. Cisplatin (DDP), a widely used anticancer agent for various types of cancers, also damages nontumor cells and tissues, which are mostly related to the activation of the inflammation pathway. Previously, we have discovered a few rational formulas of food as medicine materials that reduced systemic inflammation in in vitro and in vivo models. Hence, this study reports the ability of an optimized traditional Chinese anti-inflammatory formulation capable of synergizing the antitumor effect of DDP in vitro and in vivo. More significantly, by formulation of two anti-inflammatory herbal medicine, the Chrysanthemum × morifolium (Ramat.) Hemsl [Asteraceae] and Lonicera japonica Thunb [Caprifoliaceae] with a mediator Glycyrrhiza uralensis Fisch. ex DC [Fabaceae], a best formula relieved the kidney damage imposed by DDP. Treatments of various combinations of major chemical components of the three herbs also exhibited a similar trend for lowering the DDP-induced nephrotoxicity; however, contrary to that of the formula of herbal extracts, all chemical formulas could not recover the body weight and food intake of the tumor-bearing mice treated by DDP. Our findings suggested that the therapeutic index of DDP-based chemotherapy was able to be improved by minimizing toxicities from the two-herb formula to inhibit the inflammation in mouse tumor models and DDP-induced acute kidney injury mouse models.

LncRNA NORAD deficiency alleviates kidney injury in mice and decreases the inflammatory response and apoptosis of lipopolysaccharide-stimulated HK-2 cells via the miR-577/GOLPH3 axis

BackgroundLong noncoding RNAs (lncRNAs) are significant regulators for sepsis-associated acute kidney injury (AKI). Noncoding RNA activated by DNA damage (NORAD) is highly expressed in the serum of patients with neonatal sepsis. We aimed to reveal the role of NORAD in sepsis-associated AKI. Methods and ResultsIn this study, we established an AKI mouse model by cecal ligation and puncture (CLP) method and used the lipopolysaccharide (LPS)-stimulated HK-2 cells as the in vitro model of AKI. We identified the upregulation of NORAD expression in AKI mice and LPS-treated HK-2 cells. Silencing of NORAD alleviated renal injury by suppressing inflammation and apoptosis in vivo. The influences of NORAD suppression on cell apoptosis and inflammatory response in LPS-treated HK-2 cells were investigated by TUNEL and western blotting. NORAD deficiency inhibited HK-2 cell apoptosis and relieved the inflammation. Moreover, we explored the underlying mechanism by which NORAD regulates HK-2 cells. MiR-577 was verified to directly bind to NORAD, and GOLPH3 was identified as a target downstream miR-577. In addition, GOLPH3 overexpression countervailed the impacts of NORAD downregulation on apoptosis and inflammation in vitro. ConclusionsOur findings revealed that NORAD knockdown alleviates kidney injury in mice and decreases the inflammatory response and apoptosis of LPS-stimulated HK-2 cells via the miR-577/GOLPH3 axis.

A mouse model of exertional heatstroke-related acute kidney injury.

BackgroundAcute kidney injury (AKI) is a common complication of exertional heat stroke (EHS) with a complex pathogenesis. We established a stable mouse model of EHS-related AKI (EHS-AKI).MethodsC57BL/6 male mice were divided into 6 groups: Saline Control group, Glycerol Control group, Saline + Sham heat exercise (SHE) group, Saline + Heat exercise (HE) group, Glycerol + SHE group, and Glycerol + HE group. Samples from the Saline Control group and the Glycerol Control group were taken 6 h after the intramuscular injection of saline (4 mL/kg) or glycerol (4 mL/kg) to provide a baseline for comparisons with the other 4 groups. The other 4 groups of mice started exercise 6 h after the intramuscular injection of saline or glycerol, and were sacrificed to collect samples after exercise. Finally, serum and the pathology of kidney and muscle tissues were quantified.ResultsThere were no differences in the creatinine (Cr), blood urea nitrogen (BUN), creatine kinase (CK), and myoglobin (MYO) levels, but the interleukin 6 (IL-6) level was more increased (P<0.05) in the Glycerol Control group than the Saline Control group at the baseline. The IL-6 levels of the Glycerol + HE group were also higher than those of the Saline + HE groups at 6 and 12 h (P<0.05). The Cr levels at 12 h and 1 day, the BUN levels at 6 h, 12 h, 1 day, and 2 days in the Glycerol+ HE group were higher than the baseline levels (P<0.05). And the renal pathological scores at 6 h, 12 h, 1 day, 2 days, or 3 days were 0.79, 1.29, 1.58, 0.85, and 0.77. However, there was only slight renal pathological injury in the Saline + HE group at 12 h, and 1 day, and the scores were 0.13, and 0.41. The CK level in each group all peaked at 6 h after exercise and higher than the baseline (P<0.05). However, there was no difference in the MYO levels of each group compared to the baseline.ConclusionsWe established a stable mouse model of EHS-AKI by conducting a heat exercise after the intramuscular injection of glycerol. Our findings lay the foundation for follow-up clinical and basic research.

Sevoflurane preconditioning prevents acute renal injury caused by ischemia-reperfusion in mice via activation of the Nrf2 signaling pathway.

Oxidative stress, caused by renal ischemia reperfusion (IR)/hypoperfusion, is one of the main causes of acute kidney injury (AKI). Previous studies have demonstrated that sevoflurane (SEV) protects organs from the damage caused by oxidative stress. In the present study, mice were randomly assigned to a sham operation group (Sham), IR-vehicle group (IR+ vehicle), IR + SEV low-dose preconditioning group and an IR + SEV high-dose preconditioning group. The effect of SEV on nuclear factor E2-related factor 2 (Nrf2), a key regulatory protein of the endogenous antioxidant defense system and, consequently oxidative stress, inflammation and apoptosis-related factors, were all quantified using commercial kits or by western blotting. SEV preconditioning was demonstrated to ameliorate kidney injury as a result of decreased blood urine nitrogen and serum creatinine levels, activated Nrf2 expression in the kidney and decreased oxidative stress and inflammatory index levels an AKI mouse model. SEV preconditioning also protected injured kidney via the downregulation of caspase-3 protein expression levels. In addition, using the Nrf2 inhibitor, Brusatol, significantly abolished the SEV preconditioning renal protective effect. Using an in vitro HK-2 cell model of hypoxia/reoxygenation, it was also demonstrated that Nrf2 pathway activation was necessary for SEV to exert its beneficial effect for tubular cell injury caused by hypoxia/reoxygenation. These results indicated that SEV may protect against renal injury caused by IR via Nrf2 upregulation.

Possible kidney-lung cross-talk in COVID-19: in silico modeling of SARS-CoV-2 infection

BackgroundPublicly available genomics datasets have grown drastically during the past decades. Although most of these datasets were initially generated to answer a pre-defined scientific question, their repurposing can be useful when new challenges such as COVID-19 arise. While the establishment and use of experimental models of COVID-19 are in progress, the potential hypotheses for mechanisms of onset and progression of COVID-19 can be generated by using in silico analysis of known molecular changes during COVID-19 and targets for SARS-CoV-2 invasion.MethodsSelecting condition: COVID-19 infection leads to pneumonia and mechanical ventilation (PMV) and associated with acute kidney injury (AKI). There is increasing data demonstrating mechanistic links between AKI and lung injury caused by mechanical ventilation.Selecting targets: SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) for cell entry. We hypothesized that expression of ACE2 and TMPRSS2 would be affected in models of AKI and PMV. We therefore evaluated expression of ACE2 and TMPRSS2 as well as other novel molecular players of AKI and AKI-lung cross-talk in the publicly available microarray datasets GSE6730 and GSE60088, which represent gene expression of lungs and kidneys in mouse models of AKI and PMV, respectively.ResultsExpression of COVID-19 related genes ACE2 and TMPRSS2 was downregulated in lungs after 6 h of distant AKI effects. The expression of ACE2 decreased further after 36 h, while expression of TMPRSS2 recovered. In kidneys, both genes were downregulated by AKI, but not by distant lung injury. We also identified 53 kidney genes upregulated by PMV; and 254 lung genes upregulated by AKI, 9 genes of which were common to both organs. 3 of 9 genes were previously linked to kidney-lung cross-talk: Lcn2 (Fold Change (FC)Lung (L) = 18.6, FCKidney (K) = 6.32), Socs3 (FCL = 10.5, FCK = 10.4), Inhbb (FCL = 6.20, FCK = 6.17). This finding validates the current approach and reveals 6 new candidates, including Maff (FCL = 7.21, FCK = 5.98).ConclusionsUsing our in silico approach, we identified changes in COVID-19 related genes ACE2 and TMPRSS2 in traditional mouse models of AKI and kidney-lung cross-talk. We also found changes in new candidate genes, which could be involved in the combined kidney-lung injury during COVID-19.