Cpd-A1 alleviates acute kidney injury by inhibiting ferroptosis.

Acute kidney injury (AKI) is defined as sudden loss of renal function characterized by increased serum creatinine levels and reduced urinary output with a duration of 7 days. Ferroptosis, an iron-dependent regulated necrotic pathway, has been implicated in the progression of AKI, while ferrostatin-1 (Fer-1), a selective inhibitor of ferroptosis, inhibited renal damage, oxidative stress and tubular cell death in AKI mouse models. However, the clinical translation of Fer-1 is limited due to its lack of efficacy and metabolic instability. In this study we designed and synthesized four Fer-1 analogs (Cpd-A1, Cpd-B1, Cpd-B2, Cpd-B3) with superior plasma stability, and evaluated their therapeutic potential in the treatment of AKI. Compared with Fer-1, all the four analogs displayed a higher distribution in mouse renal tissue in a pharmacokinetic assay and a more effective ferroptosis inhibition in erastin-treated mouse tubular epithelial cells (mTECs) with Cpd-A1 (N-methyl-substituted-tetrazole-Fer-1 analog) being the most efficacious one. In hypoxia/reoxygenation (H/R)- or LPS-treated mTECs, treatment with Cpd-A1 (0.25 µM) effectively attenuated cell damage, reduced inflammatory responses, and inhibited ferroptosis. In ischemia/reperfusion (I/R)- or cecal ligation and puncture (CLP)-induced AKI mouse models, pre-injection of Cpd-A1 (1.25, 2.5, 5 mg·kg-1·d-1, i.p.) dose-dependently improved kidney function, mitigated renal tubular injury, and abrogated inflammation. We conclude that Cpd-A1 may serve as a promising therapeutic agent for the treatment of AKI.

Madecassoside alleviates acute kidney injury by regulating JNK-mediated oxidative stress and programmed cell death.

BACKGROUND: Acute kidney injury (AKI) has high morbidity and mortality, which is manifested by inflammation and apoptosis. Effective treatment methods for AKI are currently lacking. OBJECTIVE: This study demonstrated the protecting effects of Madecassoside (MA) in the cisplatin- and hypoxia-reoxygenation-induced renal tubular epithelial cells in vitro and AKI mice in vivo. METHODS: In vivo AKI mouse models were established by inducing them with cisplatin and renal ischemia-reperfusion. In vitro injury models of mouse renal tubular epithelial cells were established by inducing them with cisplatin and hypoxia and reoxygenation, respectively. The mechanism of MA effects was further explored using molecular docking and RNA-sequencing. RESULTS: MA could significantly reduce kidney injury in the cisplatin-and renal ischemia-reperfusion (IRI)-induced AKI. Further validation in the two cellular models also showed that MA had protect effects. MA can alleviate AKI in vitro and in vivo by inhibiting inflammation, cell apoptosis, and oxidative stress. MA exhibited high permeability across the Caco-2 cell, can enter cells directly. Through RNA-seq and molecular docking analysis, this study further demonstrated that MA inhibits its activity by directly binding to JNK kinase, thereby inhibiting c-JUN mediated cell apoptosis and improving AKI. In addition, MA has better renal protective effects compared to curcumin and JNK inhibitor SP600125. CONCLUSION: The results demonstrate that MA might be a potential drug for the treatment of AKI and act through the JNK/c-JUN signaling pathway.

Protein acetylation and related potential therapeutic strategies in kidney disease.

Kidney disease can be caused by various internal and external factors that have led to a continual increase in global deaths. Current treatment methods can alleviate but do not markedly prevent disease development. Further research on kidney disease has revealed the crucial function of epigenetics, especially acetylation, in the pathology and physiology of the kidney. Histone acetyltransferases (HATs), histone deacetylases (HDACs), and acetyllysine readers jointly regulate acetylation, thus affecting kidney physiological homoeostasis. Recent studies have shown that acetylation improves mechanisms and pathways involved in various types of nephropathy. The discovery and application of novel inhibitors and activators have further confirmed the important role of acetylation. In this review, we provide insights into the physiological process of acetylation and summarise its specific mechanisms and potential therapeutic effects on renal pathology.

Novel insights into STAT3 in renal diseases.

Signal transducer and activator of transcription 3 (STAT3) is a cell-signal transcription factor that has attracted considerable attention in recent years. The stimulation of cytokines and growth factors can result in the transcription of a wide range of genes that are crucial for several cellular biological processes involved in pro- and anti-inflammatory responses. STAT3 has attracted considerable interest as a result of a recent upsurge in study because of their role in directing the innate immune response and sustaining inflammatory pathways, which is a key feature in the pathogenesis of many diseases, including renal disorders. Several pathological conditions which may involve STAT3 include diabetic nephropathy, acute kidney injury, lupus nephritis, polycystic kidney disease, and renal cell carcinoma. STAT3 is expressed in various renal tissues under these pathological conditions. To better understand the role of STAT3 in the kidney and provide a theoretical foundation for STAT3-targeted therapy for renal disorders, this review covers the current work on the activities of STAT3 and its mechanisms in the pathophysiological processes of various types of renal diseases.

Production of Rhizopus oryzae lipase using optimized Yarrowia lipolytica expression system.

Yarrowia lipolytica is an alternative yeast for heterologous protein production. Based on auto-cloning vectors, a set of 18 chromogenic cloning vectors was developed, each containing one of the excisable auxotrophic selective markers URA3ex, LYS5ex, and LEU2ex, and one of six different promoters: the constitutive pTEF, the phase dependent hybrid pHp4d, and the erythritol-inducible promoters from pEYK1 and pEYL1 derivatives. These vectors allowed to increase the speed of cloning of the gene of interest. In parallel, an improved new rProt recipient strain JMY8647 was developed by abolishing filamentation and introducing an auxotrophy for lysine (Lys-), providing an additional marker for genetic engineering. Using this cloning strategy, the optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined. Among the eight targeting sequences, the SP6 signal sequence resulted in a 23% improvement in the lipase activity compared to that obtained with the wild-type ROL signal sequence. Higher specific lipase activities were obtained using hybrid erythritol-inducible promoters pHU8EYK and pEYL1-5AB, 1.9 and 2.2 times, respectively, when compared with the constitutive pTEF promoter. Two copy strains produce a 3.3 fold increase in lipase activity over the pTEF monocopy strain (266.7 versus 79.7 mU/mg).

Cpd-0225 attenuates renal fibrosis via inhibiting ALK5.

Chronic kidney disease (CKD) is an increasing public health concern, characterized by a reduced glomerular filtration rate and increased urinary albumin excretion. Renal fibrosis is an important pathological condition in patients with CKD. In this study, we evaluated the anti-fibrotic effect of Cpd-0225, a novel transforming growth factor-ß (TGF-ß) type I receptor (also known as ALK5) inhibitor, in vitro and in vivo, by comparing its effect with that of SB431542, a classic ALK5 inhibitor, which has not entered the clinical trial stage owing to multiple side effects. Our data showed that Cpd-0225 attenuated fibrotic response in TGF-ß1-stimulated human kidney tubular epithelial cells and repeated hypoxia/reoxygenation-treated mouse tubular epithelial cells. We further confirmed that Cpd-0225 improved renal tubular injury and ameliorated collagen deposition in unilateral ureteral obstruction-, ischemia/reperfusion-, and aristolochic acid-induced mouse models of renal fibrosis. In addition, molecular docking and site-directed mutagenesis showed that Cpd-0225 exerted a higher reno-protective effect than SB431542, by physically binding to the key amino acid residues, Lys232 and Lys335 of ALK5, thereby suppressing the phosphorylation of Smad3 and ERK1/2. Taken together, these findings suggest that Cpd-0225 administration attenuates renal fibrosis via ALK5-dependent mechanisms and displays a more effective therapeutic effect than SB431542. Thus, Cpd-0225 may serve as a potential therapeutic agent for the treatment of CKD.

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.

Investigation of Structural Features of Two Related Lipases and the Impact on Fatty Acid Specificity in Vegetable Fats.

One of the indispensable applications of lipases in modification of oils and fats is the possibility to tailor the fatty acid content of triacylglycerols (TAGs), to meet specific requirements from various applications in food, nutrition, and cosmetic industries. Oleic acid (C18:1) and stearic acid (C18:0) are two common long fatty acids in the side chain of triglycerides in plant fats and oils that have similar chemical composition and structures, except for an unsaturated bond between C9 and C10 in oleic acid. Two lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL), show activity in reactions involving oleate and stearate, and share high sequence and structural identity. In this research, the preference for one of these two similar fatty acid side chains was investigated for the two lipases and was related to the respective enzyme structure. From transesterification reactions with 1:1 (molar ratio) mixed ethyl stearate (ES) and ethyl oleate (EO), both RML and ROL showed a higher activity towards EO than ES, but RML showed around 10% higher preference for ES compared with ROL. In silico results showed that stearate has a less stable interaction with the substrate binding crevice in both RML and ROL and higher tendency to freely move out of the substrate binding region, compared with oleate whose structure is more rigid due to the existence of the double bond. However, Trp88 from RML which is an Ala at the identical position in ROL shows a significant stabilization effect in the substrate interaction in RML, especially with stearate as a ligand.

Insulin-like growth factor binding protein 7 promotes acute kidney injury by alleviating poly ADP ribose polymerase 1 degradation.

The novel biomarker, insulin-like growth factor binding protein 7 (IGFBP7), is used clinically to predict different types of acute kidney injury (AKI) and has drawn significant attention as a urinary biomarker. However, as a secreted protein in the circulation of patients with AKI, it is unclear whether IGFBP7 acts as a key regulator in AKI progression, and if mechanisms underlying its upregulation still need to be determined. Here we found that IGFBP7 is highly expressed in the blood and urine of patients and mice with AKI, possibly via a c-Jun-dependent mechanism, and is positively correlated with kidney dysfunction. Global knockout of IGFBP7 ameliorated kidney dysfunction, inflammatory responses, and programmed cell death in murine models of cisplatin-, kidney ischemia/reperfusion-, and lipopolysaccharide-induced AKI. IGFBP7 mainly originated from kidney tubular epithelial cells. Conditional knockout of IGFBP7 from the kidney protected against AKI. By contrast, rescue of IGFBP7 expression in IGFBP7-knockout mice restored kidney damage and inflammation. IGFBP7 function was determined in vitro using recombinant IGFBP7 protein, IGFBP7 knockdown, or overexpression. Additionally, IGFBP7 was found to bind to poly [ADP-ribose] polymerase 1 (PARP1) and inhibit its degradation by antagonizing the E3 ubiquitin ligase ring finger protein 4 (RNF4). Thus, IGFBP7 in circulation acts as a biomarker and key mediator of AKI by inhibiting RNF4/PARP1-mediated tubular injury and inflammation. Hence, over-activation of the IGFBP7/PARP1 axis represents a promising target for AKI treatment.

Identification of a Catalytic Nucleophile-Activating Network in the endo-α-N-Acetylgalactosaminidase of Family GH101.

Bifidobacterium longum endo-α-N-acetylgalactosaminidase (GH101), EngBF, is highly specific toward the mucin Core 1 glycan, Galß1-3GalNAc. Apart from the side chains involved in the retaining mechanism of EngBF, Asp-682 is important for the activity. In the crystal structures of both EngBF and EngSP (from Streptococcus pneumoniae), we identified a conserved water molecule in proximity to Asp-682 and the homologue residue in EngSP. The water molecule also coordinates the catalytic nucleophile and three other residues conserved in GH101 enzymes; in EngBF, these residues are His-685, His-718, and Asn-720. With casein-glycomacropeptide as the substrate, the importance of Asp-682 was confirmed by the lack of a detectable activity for the D682N enzyme. The enzyme variants, H685A, H718A, H685Q, and H718Q, all displayed only a modestly reduction in kcat of up to 15 fold for the H718A variant. However, the double-substituted variants, H685A/H718A and H685Q/H718Q, had a greatly reduced kcat value by about 200 fold compared to that of wild-type EngBF. With the synthetic substrate, Galß(1-3)GalNAcα1-para-nitrophenol, kcat of the double-substituted variants was only up to 30-fold reduced and was found to increase with pH. Compared to the pre-steady-state kinetics of wild-type EngBF, a burst of about the size of the enzyme concentration was absent with the double-substituted EngBF variants, indicating that the nucleophilic attack had become at least as slow as the hydrolysis of the enzyme intermediate. Together, the results indicate that not only Asp-682 but also the entire conserved network of His-685, His-718, and what we suggest is a catalytic water molecule is important in the activation of the catalytic nucleophile.