CCDC92 promotes podocyte injury by regulating PA28α/ABCA1/cholesterol efflux axis in type 2 diabetic mice.

Podocyte lipotoxicity mediated by impaired cellular cholesterol efflux plays a crucial role in the development of diabetic kidney disease (DKD), and the identification of potential therapeutic targets that regulate podocyte cholesterol homeostasis has clinical significance. Coiled-coil domain containing 92 (CCDC92) is a novel molecule related to metabolic disorders and insulin resistance. However, whether the expression level of CCDC92 is changed in kidney parenchymal cells and the role of CCDC92 in podocytes remain unclear. In this study, we found that Ccdc92 was significantly induced in glomeruli from type 2 diabetic mice, especially in podocytes. Importantly, upregulation of Ccdc92 in glomeruli was positively correlated with an increased urine albumin-to-creatinine ratio (UACR) and podocyte loss. Functionally, podocyte-specific deletion of Ccdc92 attenuated proteinuria, glomerular expansion and podocyte injury in mice with DKD. We further demonstrated that Ccdc92 contributed to lipid accumulation by inhibiting cholesterol efflux, finally promoting podocyte injury. Mechanistically, Ccdc92 promoted the degradation of ABCA1 by regulating PA28α-mediated proteasome activity and then reduced cholesterol efflux. Thus, our studies indicate that Ccdc92 contributes to podocyte injury by regulating the PA28α/ABCA1/cholesterol efflux axis in DKD.

Targeting RNA oxidation by ISG20-mediated degradation is a potential therapeutic strategy for acute kidney injury.

Oxidative stress plays a central role in the pathophysiology of acute kidney injury (AKI). Although RNA is one of the most vulnerable cell components to oxidative damage, it is unclear whether RNA oxidation is involved in the pathogenesis of AKI. In this study, we found that the level of RNA oxidation was significantly enhanced in kidneys of patients with acute tubular necrosis (ATN) and in the renal tubular epithelial cells (TECs) of mice with AKI, and oxidized RNA overload resulted in TEC injury. We further identified interferon-stimulated gene 20 (ISG20) as a novel regulator of RNA oxidation in AKI. Tubule-specific deficiency of ISG20 significantly aggravated renal injury and RNA oxidation in the ischemia/reperfusion-induced AKI mouse model and ISG20 restricted RNA oxidation in an exoribonuclease activity-dependent manner. Importantly, overexpression of ISG20 protected against oxidized RNA overproduction and renal ischemia/reperfusion injury in mice and ameliorated subsequent protein aggresome accumulation, endoplasmic reticulum stress, and unfolded protein response. Thus, our findings provide direct evidence that RNA oxidation contributes to the pathogenesis of AKI and that ISG20 importantly participates in the degradation of oxidized RNA, suggesting that targeting ISG20-handled RNA oxidation may be an innovative therapeutic strategy for AKI.

Histone deacetylase 9 exacerbates podocyte injury in hyperhomocysteinemia through epigenetic repression of Klotho.

Although hyperhomocysteinemia (hHcys) has been recognized as an important independent risk factor in the progression of end-stage renal disease and the development of cardiovascular complications related to end-stage renal disease, the mechanisms triggering pathogenic actions of hHcys are not fully understood. The present study was mainly designed to investigate the role of HDACs in renal injury induced by hHcys. Firstly, we identified the expression patterns of HDACs and found that, among zinc-dependent HDACs, HDAC9 was preferentially upregulated in the kidney from mice with hHcys. Deficiency or pharmacological inhibition of HDAC9 ameliorated renal injury in mice with hHcys. Moreover, podocyte-specific deletion of HDAC9 significantly attenuated podocyte injury and proteinuria. In vitro, gene silencing of HDAC9 attenuated podocyte injury by inhibiting apoptosis, reducing oxidative stress and maintaining the expressions of podocyte slit diaphragm proteins. Mechanically, we proved for the first time that HDAC9 reduced the acetylation level of H3K9 in the promoter of Klotho, then inhibited gene transcription of Klotho, finally aggravating podocyte injury in hHcys. In conclusion, our results indicated that targeting of HDAC9 might be an attractive therapeutic strategy for the treatment of renal injury induced by hHcys.

Targeting tissue-resident memory CD8+ T cells in the kidney is a potential therapeutic strategy to ameliorate podocyte injury and glomerulosclerosis.

Although tissue-resident-memory T (TRM) cells, a recently identified non-circulating memory T cell population, play a crucial role in mediating local immune responses and protect against pathogens upon local reinfection, the composition, effector function, and specificity of TRM cells in the kidney and their relevance for chronic kidney disease remain unknown. In this study, we found that renal tissue displayed high abundance of tissue-resident lymphocytes, and the proportion of CD8+ TRM cells was significantly increased in the kidney from patients and mice with focal segmental glomerulosclerosis (FSGS), diabetic kidney disease (DKD), and lupus nephritis (LN). Mechanistically, IL-15 significantly promoted CD8+ TRM cell formation and activation, thereby promoting podocyte injury and glomerulosclerosis. Interestingly, Sparsentan, the dual angiotensin II (Ang II) receptor and endothelin type A receptor antagonist, can also reduce TRM cell responses by intervening IL-15 signaling, exploring its new pharmacological functions. Mechanistically, Sparsentan inhibited Ang II or endothelin-1 (ET-1)-mediated IL-15 signaling, thereby further regulating renal CD8+ TRM cell fates. Collectively, our studies provide direct evidence for the pivotal role of renal CD8+ TRM cells in podocyte injury and further strengthen that targeting TRM cells represents a novel therapeutic strategy for patients with glomerular diseases.

Morphological Profiling Identifies the Motor Protein Eg5 as Cellular Target of Spirooxindoles.

Oxindoles and iso-oxindoles are natural product-derived scaffolds that provide inspiration for the design and synthesis of novel biologically relevant compound classes. Notably, the spirocyclic connection of oxindoles with iso-oxindoles has not been explored by nature but promises to provide structurally related compounds endowed with novel bioactivity. Therefore, methods for their efficient synthesis and the conclusive discovery of their cellular targets are highly desirable. We describe a selective RhIII -catalyzed scaffold-divergent synthesis of spirooxindole-isooxindoles and spirooxindole-oxindoles from differently protected diazooxindoles and N-pivaloyloxy aryl amides which includes a functional group-controlled Lossen rearrangement as key step. Unbiased morphological profiling of a corresponding compound collection in the Cell Painting assay efficiently identified the mitotic kinesin Eg5 as the cellular target of the spirooxindoles, defining a unique Eg5 inhibitor chemotype.

Identification of Dihydroorotate Dehydrogenase Inhibitors Using the Cell Painting Assay.

Profiling approaches have been increasingly employed for the characterization of disease-relevant phenotypes or compound perturbation as they provide a broad, unbiased view on impaired cellular states. We report that morphological profiling using the cell painting assay (CPA) can detect modulators of de novo pyrimidine biosynthesis and of dihydroorotate dehydrogenase (DHODH) in particular. The CPA can differentiate between impairment of pyrimidine and folate metabolism, which both affect cellular nucleotide pools. The identified morphological signature is shared by inhibitors of DHODH and the functionally tightly coupled complex III of the mitochondrial respiratory chain as well as by UMP synthase, which is downstream of DHODH. The CPA appears to be particularly suited for the detection of DHODH inhibitors at the site of their action in cells. As DHODH is a validated therapeutic target, the CPA will enable unbiased identification of DHODH inhibitors and inhibitors of de novo pyrimidine biosynthesis for biological research and drug discovery.

Chemical Biology Tools for Protein Lysine Acylation.

Lysine acylation plays pivotal roles in cell physiology, including DNA transcription and repair, signal transduction, immune defense, metabolism, and many other key cellular processes. Molecular mechanisms of dysregulated lysine acylation are closely involved in the pathophysiological progress of many human diseases, most notably cancers. In recent years, chemical biology tools have become instrumental in studying the function of post-translational modifications (PTMs), identifying new "writers", "erasers" and "readers", and in targeted therapies. Here, we describe key developments in chemical biology approaches that have advanced the study of lysine acylation and its regulatory proteins (2016-2021). We further discuss the discovery of ligands (inhibitors and PROTACs) that are capable of targeting regulators of lysine acylation. Next, we discuss some current challenges of these chemical biology probes and suggest how chemists and biologists can utilize chemical probes with more discriminating capacity. Finally, we suggest some critical considerations in future studies of PTMs from our perspective.

Natural STAT3 inhibitors: A mini perspective.

Signal transducer and activator of transcription 3 (STAT3) plays pivotal role in several cellular processes such as cell proliferation and survival and has been found to be aberrantly activated in many cancers. STAT3 is largely believed to be one of the key oncogenes and crucial therapeutic targets. Much research has suggested the leading mechanisms for regulating the STAT3 pathway and its role in promoting tumorigenesis. Therefore, intensive efforts have been devoted to develop potent STAT3 inhibitors and several of them are currently undergoing clinical trials. Nevertheless, many natural products were identified as STAT3 inhibitors but attract less attention compared to the small molecule counterpart. In this review, the development of natural STAT3 inhibitors with an emphasis on their biological profile and chemical synthesis are detailed. The current state of STAT3 inhibitors and the future directions and opportunities for STAT3 inhibitor are discussed.

Chemical Probes Reveal Sirt2's New Function as a Robust "Eraser" of Lysine Lipoylation.

Lysine lipoylation, a highly conserved lysine post-translational modification, plays a critical role in regulating cell metabolism. The catalytic activity of a number of vital metabolic proteins, such as pyruvate dehydrogenase (PDH), depends on lysine lipoylation. Despite its important roles, the detailed biological regulatory mechanism of lysine lipoylation remains largely unexplored. Herein we designed a powerful affinity-based probe, KPlip, to interrogate the interactions of lipoylated peptide/proteins under native cellular environment. Large-scale chemical proteomics analysis revealed a number of binding proteins of KPlip, including sirtuin 2 (Sirt2), an NAD+-dependent protein deacylase. To explore the potential activity of Sirt2 toward lysine lipoylation, we designed a single-step fluorogenic probe, KTlip, which reports delipoylation activity in a continuous manner. The results showed that Sirt2 led to significant delipoylation of KTlip, displaying up to a 60-fold fluorescence increase in the assay. Further kinetic experiments with different peptide substrates revealed that Sirt2 can catalyze the delipoylation of peptide (DLAT-PDH, K259) with a remarkable catalytic efficiency (kcat/Km) of 3.26 × 103 s-1 M-1. The activity is about 400-fold higher than that of Sirt4, the only mammalian enzyme with known delipoylation activity. Furthermore, overexpression and silencing experiments demonstrated that Sirt2 regulates the lipoylation level and the activity of endogenous PDH, thus unequivocally confirming that PDH is a genuine physiological substrate of Sirt2. Using our chemical probes, we have successfully established the relationship between Sirt2 and lysine lipoylation in living cells for the first time. We envision that such chemical probes will serve as useful tools for delineating the roles of lysine lipoylation in biology and diseases.

Fluorescent Probes for Single-Step Detection and Proteomic Profiling of Histone Deacetylases.

Histone deacetylases (HDACs) play important roles in regulating various physiological and pathological processes. Developing fluorescent probes capable of detecting HDAC activity can help further elucidate the roles of HDACs in biology. In this study, we first developed a set of activity-based fluorescent probes by incorporating the Kac residue and the O-NBD group. Upon enzymatic removal of the acetyl group in the Kac residue, the released free amine reacted intramolecularly with the O-NBD moiety, resulting in turn-on fluorescence. These designed probes are capable of detecting HDAC activity in a continuous fashion, thereby eliminating the extra step of fluorescence development. Remarkably, the amount of turn-on fluorescence can be as high as 50-fold, which is superior to the existing one-step HDAC fluorescent probes. Inhibition experiments further proved that the probes can serve as useful tools for screening HDAC inhibitors. Building on these results, we moved on and designed a dual-purpose fluorescent probe by introducing a diazirine photo-cross-linker into the probe. The resulting probe was not only capable of reporting enzymatic activity but also able to directly identify and capture the protein targets from the complex cellular environment. By combining a fluorometric method and in-gel fluorescence scanning technique, we found that epigenetic readers and erasers can be readily identified and differentiated using a single probe. This is not achievable with traditional photoaffinity probes. In light of the prominent properties and the diverse functions of this newly developed probe, we envision that it can provide a robust tool for functional analysis of HDACs and facilitate future drug discovery in epigenetics.

Fluorescent probes for detecting monoamine oxidase activity and cell imaging.

A series of new fluorogenic probes for monoamine oxidases (MAOs) were reported based on an oxidation and ß-elimination mechanism. The limits of detection of the probes for MAO-A and -B were determined to be 3.5 and 6.0 µg mL(-1) respectively. These probes displayed strong activity towards MAOs, especially MAO-B. Cellular imaging studies were also successfully conducted with MCF-7 cells.

Development and evaluation of a second victim training course for nursing managers.

BACKGROUND: Most nursing managers are not fully aware of second victims and may not be able to provide support. Moreover, there are relatively few training courses for nursing managers about second victims. AIM: To describe the construction and evaluation of a second victim course for nursing managers. DESIGN: A single-group pretest-posttest study design was used. SETTING: A large comprehensive tertiary hospital with over 3000 beds in China. PARTICIPANTS: Forty-nine nursing managers who met the inclusion and exclusion criteria participated in this training. Sixteen clinical frontline nurses who experienced adverse events within three months following the training were also invited. METHODS: The course "Second Victim & Empathy Communication" was developed through a literature review and expert consultation and consisted of 4 unit modules: (1) adverse events & second victims, (2) the recovery trajectory of second victims, (3) second victim supportive resources, and (4) key strategies of empathy communication. A course evaluation questionnaire, an empathy communication questionnaire for nursing managers, a second victim evaluation questionnaire, and an open-ended question were used to measure the feasibility, acceptability and effectiveness of the course. RESULTS: >97.96 % of the nursing managers were satisfied with the course, >97.96 % had learned new knowledge, and >95.92 % had changed their behavior and attitudes toward second victims. Their levels of empathetic communication differed significantly before and after training (t = -2.170, P = 0.035). Among these nursing managers, twenty-six participants provided positive and meaningful feedback and suggestions to the course by answering an open-ended question. A total of 66.6 % to 100 % of second victims were satisfied with the empathetic communication behavior exhibited by nursing managers. CONCLUSION: The second victim training course is feasible and can be used for clinical training to enhance nursing managers' understanding of second victims and enhance their empathetic communication.

CCDC92 deficiency ameliorates podocyte lipotoxicity in diabetic kidney disease.

BACKGROUND AND AIMS: Podocyte injury is considered as the most important early event contributing to diabetic kidney disease (DKD). Recent findings provide new insights into the roles of lipids and lipid-modulating proteins as key determinants of podocyte function in health and kidney disease. CCDC92, a novel member of coiled-coil domain-containing protein family, was indicated relevant to lipid metabolism, coronary heart disease and type 2 diabetes. However, the expression pattern and role of CCDC92 in the kidney is not clear. This study was designed to elucidate the contribution of CCDC92 in the pathogenesis of DKD. METHODS: Sections with a pathological diagnosis of different classes of DKD, including subjects with mild DKD (class II, n = 6), subjects with moderate DKD (class III, n = 6) or subjects with severe DKD (class IV, n = 6), and control samples (n = 12) were detected for the expression level of CCDC92 and lipid accumulation. Two types of diabetic mice model (db/db and HFD/STZ) in podocyte-specific Ccdc92 knockout background were generated to clarify the role of CCDC92 in podocyte lipotoxicity. RESULTS: The level of CCDC92 was increased in renal biopsies sections from patients with DKD, which was correlated with eGFR and lipid accumulation in glomeruli. In animal studies, CCDC92 were also induced in the kidney from two independent diabetic models, especially in podocytes. Podocyte-specific deletion of Ccdc92 ameliorated podocyte injury and ectopic lipid deposition under diabetic condition. Mechanically, CCDC92 promoted podocyte lipotoxicity, at least in part through ABCA1 signaling-mediated lipid homeostasis. CONCLUSION: Our studies demonstrates that CCDC92 acts as a novel regulator of lipid homeostasis to promote podocyte injury in DKD, suggesting that CCDC92 might be a potential biomarker of podocyte injury in DKD, and targeting CCDC92 may be an effective innovative therapeutic strategy for patients with DKD.

Nickel-catalyzed C-H direct amination of benzoxazoles with secondary amines.

In this article, a facile, efficient and practical method for Ni-catalyzed direct C-H amination of benzoxazole with secondary amines has been developed. This procedure requires Ni(OAc)(2)·4H(2)O as catalyst, TBHP as oxidant and acid as the additive. A variety of substituted benzoxazol-2-amines were synthesized in moderate to good yields.

FGFR2-BRD4 Axis Regulates Transcriptional Networks of Histone 3 Modification and Synergy Between Its Inhibitors and PD-1/PD-L1 in a TNBC Mouse Model.

Epigenetic reprogramming is an independent mode of gene expression that often involves changes in the transcription and chromatin structure due to tumor initiation and development. In this study, we developed a specifically modified peptide array and searched for a recognized epigenetic reader. Our results demonstrated that BRD4 is not only an acetylation reader but of propionylation as well. We also studied the quantitative binding affinities between modified peptides and epigenetic regulators by isothermal titration calorimetry (ITC). Furthermore, we introduced the Fgfr2-S252W transgenic mouse model to confirm that this acetylation is associated with the activation of c-Myc and drives tumor formation. Targeted disruption of BRD4 in Fgfr2-S252W mouse tumor cells also confirmed that BRD4 is a key regulator of histone 3 acetylation. Finally, we developed a tumor slice culture system and demonstrated the synergy between immune checkpoint blockade and targeted therapy in triple-negative breast cancer (TNBC). These data extend our understanding of epigenetic reprogramming and epigenetics-based therapies.

GPR87 promotes renal tubulointerstitial fibrosis by accelerating glycolysis and mitochondrial injury.

Renal tubulointerstitial fibrosis is the hallmark of chronic kidney disease (CKD) and the best predictor of renal survival. However, current treatments for CKD remain extremely limited. Therefore, novel therapeutic targets are urgently needed to either stop or reverse CKD progression. The present study was designed to explore the potential role of GPR87, a member of the G protein-coupled receptors (GPCRs) family, in the pathogenesis of tubulointerstitial fibrosis. It was found that GPR87 was significantly induced in the kidney, especially in tubular areas, from different mouse models of renal fibrosis, including unilateral ureteral obstruction (UUO) nephropathy, aristolochic acid nephropathy, and diabetic nephropathy, respectively. Tubule-specific GPR87 deletion dramatically ameliorated tubulointerstitial fibrosis in UUO mice. Mechanistically, GPR87 accelerated glycolysis and mitochondrial injury by YAP-hexokinase-2 signaling, thereby promoting renal fibrosis. Importantly, the upregulation of GPR87 was also found in the kidney from patients with various CKD, indicating that the induction of GPR87 may be a common feature of human kidney diseases. Collectively, our studies for the first time demonstrate that GPR87 plays a pivotal role in renal fibrosis at least in part by accelerating glycolysis and mitochondrial injury, suggesting that targeting GPR87 may represent a novel therapeutic strategy for patients with CKD.

JAML promotes acute kidney injury mainly through a macrophage-dependent mechanism.

Although macrophages are undoubtedly attractive therapeutic targets for acute kidney injury (AKI) because of their critical roles in renal inflammation and repair, the underlying mechanisms of macrophage phenotype switching and efferocytosis in the regulation of inflammatory responses during AKI are still largely unclear. The present study elucidated the role of junctional adhesion molecule-like protein (JAML) in the pathogenesis of AKI. We found that JAML was significantly upregulated in kidneys from 2 different murine AKI models including renal ischemia/reperfusion injury (IRI) and cisplatin-induced AKI. By generation of bone marrow chimeric mice, macrophage-specific and tubular cell-specific Jaml conditional knockout mice, we demonstrated JAML promoted AKI mainly via a macrophage-dependent mechanism and found that JAML-mediated macrophage phenotype polarization and efferocytosis is one of the critical signal transduction pathways linking inflammatory responses to AKI. Mechanistically, the effects of JAML on the regulation of macrophages were, at least in part, associated with a macrophage-inducible C-type lectin-dependent mechanism. Collectively, our studies explore for the first time to our knowledge new biological functions of JAML in macrophages and conclude that JAML is an important mediator and biomarker of AKI. Pharmacological targeting of JAML-mediated signaling pathways at multiple levels may provide a novel therapeutic strategy for patients with AKI.

Copper-catalyzed direct oxidative C-H amination of benzoxazoles with formamides or secondary amines under mild conditions.

A facile, efficient, and practical method for copper-catalyzed direct C-H amination of benzoxazoles with formamides or secondary amines has been developed. The system can be performed in the absence of external base and only requires O(2) or even air as oxidant. A variety of substituted benzoxazol-2-amines were synthesized with moderate to excellent yield.

Synthesis and fluorescence properties of red-to-near-infrared-emitting push-pull dyes based on benzodioxazole scaffolds.

Fluorescence imaging with high temporal and spatial resolution has emerged as one of the most promising techniques to monitor biomolecules and biological processes in living systems. Among many kinds of small molecular fluorescent dyes, 2,1,3-benzoxadiazole (BD) derivatives have been widely applied in many chemical and biological applications due to their excellent photophysical properties. However, only a limited number of BD dyes with long emission wavelengths were reported. Herein, we have reported a new class of red-to near-infrared-emitting small molecular dyes 2a-3a based on benzodioxazole scaffolds, which are named VBDfluors. To bathochromically shift both absorption and emission, the conjugation system was extended by introducing electron-withdrawing group-substituted vinyl groups at position 7 via a Knoevenagel condensation reaction. The basic photophysical properties of VBDfluors were detected and summarized. The VBDfluors display excellent photophysical properties, including emission in the red-to-NIR region, large Stokes shifts, good stability/photostability and cell permeability. The geometry of the molecules was optimized by density functional theory (DFT) and time-dependent DFT (TDDFT) methods. Bioimaging results indicated that 2a and 3a exhibited excellent cell permeability and could be utilized for visualization of lipid droplets in living cells.

Colorimetric and Fluorescent Dual-Signal Chemosensor for Lysine and Arginine and Its Application to Detect Amines in Solid-Phase Peptide Synthesis.

Lysine (Lys) and arginine (Arg), as two of the most alkaline amino acids among 20 common amino acids, are closely involved in many vital biological processes and biomaterial synthesis. Abnormal levels of Lys and Arg can lead to various diseases. Although a limited number of fluorescent probes for Lys and Arg have been reported, many of them are not sensitive enough due to the moderate fluorescence signal and on-off mode. In addition, none of them were applied for detecting amine groups in solid-phase peptide synthesis. In this study, we designed and synthesized optical fluorescent probe 1 based on the benzoxadiazole fluorophore, which could undergo an accelerated hydrolysis reaction under basic conditions. Probe 1 revealed excellent selectivity toward alkaline Lys and Arg over other common amino acids with both fluorometric and colorimetric readouts. After treatment with Lys and Arg, probe 1 could emit a turn-on fluorescent response at 580 nm with a distinct color change from pink to yellow. The limit of detection for Lys and Arg was calculated to be 1.1 and 1.39 µM, respectively. We also successfully applied probe 1 for the visualization of Arg in living cells. Moreover, to the best of our knowledge, probe 1 provided the first fluorescent platform to detect -NH2 groups in solid-phase synthesis of peptides with distinct fluorescent and colorimetric changes. We envision that the probe can provide an alternative method for the traditional Kaiser test.