Discovery of a SUCNR1 antagonist for potential treatment of diabetic nephropathy: In silico and in vitro studies.

Diabetic nephropathy (DN) is one of the most severe complications of diabetes mellitus. Succinate Receptor 1 (SUCNR1), a member of the G-protein-coupled receptor (GPCR) family, represents a potential target for treatment of DN. Here, utilizing multi-strategy in silico virtual screening methods containing AlphaFold2 modelling, molecular dynamics (MD) simulation, ligand-based pharmacophore screening, molecular docking and machine learning-based similarity clustering, we successfully identified a novel antagonist of SUCNR1, AK-968/12117473 (Cpd3). Through extensive in vitro experiments, including dual-luciferase reporter assay, cellular thermal shift assay, immunofluorescence, and western blotting, we substantiated that Cpd3 could specifically target SUCNR1, inhibit the activation of NF-κB pathway, and ameliorate epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in renal tubular epithelial cells (NRK-52E) under high glucose conditions. Further in silico simulations revealed the molecular basis of the SUCNR1-Cpd3 interaction, and the in vitro metabolic stability assay indicated favorable drug-like pharmacokinetic properties of Cpd3. This work not only successfully pinpointed Cpd3 as a specific antagonist of SUCNR1 to serve as a promising candidate in the realm of therapeutic interventions for DN, but also provides a paradigm of dry-wet combined discovery strategies for GPCR-based therapeutics.

The degradation of TGR5 mediated by Smurf1 contributes to diabetic nephropathy.

The multiple roles of TGR5 in the regulation of glucose metabolism, inflammation, and oxidative stress have drawn attention as therapeutic candidates for diabetes-related kidney disease. However, diabetes induces downregulation of renal TGR5 protein expression, and the regulatory mechanisms have not been clarified. Here, we identify that Smurf1, an E3 ubiquitin ligase, is a critical interactor of TGR5 and mediates the ubiquitination and proteasomal degradation of TGR5 under high glucose stimulation in glomerular mesangial cells. Genetic deficiency of Smurf1 restores TGR5 protein expression and attenuates renal injuries in diabetic mice. Mechanistically, Smurf1 interacts with the TGR5 ICL2 region by its HECT domain and induces K11/K48-linked polyubiquitination of TGR5 at K306 residue. Moreover, restoration of TGR5 protects db/db mice from diabetic nephropathy. These observations elucidate the critical role of Smurf1 in regulating TGR5 stability, suggesting that pharmacological targeting of the interaction between Smurf1 and TGR5 could serve as a promising therapeutic strategy against diabetic nephropathy.

Connexin32 Promotes the Activation of Foxo3a to Ameliorate Diabetic Nephropathy via Inhibiting the Polyubiquitination and Degradation of Sirt1.

Aims: Renal oxidative stress (OSS) is the leading cause of diabetic nephropathy (DN). The silent information regulator 1/forkhead boxo3a (Sirt1/Foxo3a) pathway plays an essential role in regulating the antioxidant enzyme system. In this study, we aimed to investigate the mechanism of connexin32 (Cx32) on the antioxidant enzyme system in DN. Results: In this study, Cx32 overexpression significantly reduced reactive oxygen species generation and effectively inhibited the excessive production of extracellular matrix such as fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in high-glucose (HG)-induced glomerular mesangial cells. In addition, Cx32 overexpression reversed the downregulation of Sirt1, and promoted the nuclear transcription of Foxo3a, subsequently activating the antioxidant enzymes including catalase and manganese superoxide dismutase (MnSOD), however, Cx32 knockdown showed the opposite effects. A further mechanism study showed that Cx32 promoted the autoubiquitination and degradation of Smad ubiquitylation regulatory factor-1 (Smurf1), thereby reducing the ubiquitination of Sirt1 at Lys335 and the degradation of Sirt1. Moreover, the in vivo results showed that adenovirus-mediated Cx32 overexpression activated the Sirt1/Foxo3a pathway, and inhibited OSS in the kidney tissues, eventually improving the renal function and glomerulosclerosis in diabetic mice. Innovation: This study highlighted the antioxidant role of Cx32-Sirt1-Foxo3a axis to alleviate DN, which is a new mechanism of Cx32 alleviating DN. Conclusion: Cx32 alleviated DN via activating the Sirt1/Foxo3a antioxidant pathway. The specific mechanism was that Cx32 upregulated the Sirt1 expression through reducing the ubiquitination of Lys335 of Sirt1 by inhibiting Smurf1. Antioxid. Redox Signal. 39, 241-261.

Fyn deficiency inhibits oxidative stress by decreasing c-Cbl-mediated ubiquitination of Sirt1 to attenuate diabetic renal fibrosis.

OBJECTIVE: Oxidative stress (OS) is the main cause leading to diabetic renal fibrosis. Recently, Fyn was paid much attention on OS and emerged as a pivotal player in acute kidney injury, while whether Fyn regulates oxidative stress in chronic diabetes nephropathy (DN) has not been clarified yet. The purpose of this study was to identify the role of Fyn in DN and elucidated its regulatory mechanism. METHODS: The db/db mice and littermate control C57BKS/J mice were injected by tail vein with Fyn interfering adenovirus or Fyn overexpressing adenovirus to investigate the role of Fyn in vivo. Primary glomerular mesangial cells (GMCs) were used for in vitro studies. RESULTS: Fyn was up-regulated in high glucose (HG)-induced GMCs and kidneys of diabetic mice. Additionally, Fyn knockdown reduced the level of OS in HG-induced GMCs and kidneys of diabetic mice, thereby ameliorating diabetic renal fibrosis. While overexpression of Fyn significantly increased the level of OS in GMCs and kidney tissues, resulting in renal damage. Moreover, Fyn deficiency exerted antioxidant effects by activating the Sirt1/Foxo3a pathway. Mechanistically, Fyn facilitated the combination of c-Cbl and Sirt1 by phosphorylating c-Cbl at Tyr731, which triggered K48-linked polyubiquitination of Sirt1 at Lys377 and Lys513 by c-Cbl and promoted Sirt1 degradation, impairing the antioxidant effects of Foxo3a. CONCLUSIONS: Fyn deficiency promoted Foxo3a nuclear transcription via reducing the ubiquitination of Sirt1 by c-Cbl, thereby alleviating renal oxidative damage in diabetic mice. These results identified Fyn as a potential therapeutic target against DN.

The ubiquitination of CKIP-1 mediated by Src aggravates diabetic renal fibrosis (original article).

Renal chronic inflammation is an important hallmark of diabetic renal fibrosis. Casein kinase 2 interacting protein 1 (CKIP-1) performs a nephroprotective role in the pathogenesis of diabetic nephropathy (DN), which is dramatically decreased in diabetic kidneys. However, whether CKIP-1 regulates inflammation to ameliorate renal fibrosis remains unclear and it is interesting to clarify the degradation mechanism of CKIP-1. Here, we identified CKIP-1 expression was down-regulated in diabetic kidneys and knockout (KO) of CKIP-1 increased c-Jun expression and extra cellular matrix (ECM) in kidneys of normal mice, and knockout (KO) of CKIP-1 further exacerbated renal inflammatory fibrosis in diabetic mice. Moreover, the activated Src kinase interacted with CKIP-1 at Lys252 and increased K48 linked polyubiquitination and proteasome degradation of CKIP-1 in HG induced GMCs and diabetic kidneys. Mechanistically, Src facilitating the binding of c-Cbl with CKIP-1 by promoting the phosphorylation of c-Cbl, thereby increasing Cbl-mediated ubiquitination of CKIP-1 to down-regulate CKIP-1 protein expression. Thus, our study highlighted the anti-inflammation role of CKIP-1 and clarified the mechanism of CKIP-1 degradation in DN.

Gentiopicroside targets PAQR3 to activate the PI3K/AKT signaling pathway and ameliorate disordered glucose and lipid metabolism.

The obstruction of post-insulin receptor signaling is the main mechanism of insulin-resistant diabetes. Progestin and adipoQ receptor 3 (PAQR3), a key regulator of inflammation and metabolism, can negatively regulate the PI3K/AKT signaling pathway. Here, we report that gentiopicroside (GPS), the main bioactive secoiridoid glycoside of Gentiana manshurica Kitagawa, decreased lipid synthesis and increased glucose utilization in palmitic acid (PA) treated HepG2 cells. Additionally, GPS improved glycolipid metabolism in streptozotocin (STZ) treated high-fat diet (HFD)-induced diabetic mice. Our findings revealed that GPS promoted the activation of the PI3K/AKT axis by facilitating DNA-binding protein 2 (DDB2)-mediated PAQR3 ubiquitinated degradation. Moreover, results of surface plasmon resonance (SPR), microscale thermophoresis (MST) and thermal shift assay (TSA) indicated that GPS directly binds to PAQR3. Results of molecular docking and cellular thermal shift assay (CETSA) revealed that GPS directly bound to the amino acids of the PAQR3 NH2-terminus including Leu40, Asp42, Glu69, Tyr125 and Ser129, and spatially inhibited the interaction between PAQR3 and the PI3K catalytic subunit (P110α) to restore the PI3K/AKT signaling pathway. In summary, our study identified GPS, which inhibits PAQR3 expression and directly targets PAQR3 to restore insulin signaling pathway, as a potential drug candidate for the treatment of diabetes.

Fraxin Promotes the Activation of Nrf2/ARE Pathway via Increasing the Expression of Connexin43 to Ameliorate Diabetic Renal Fibrosis.

Diabetic nephropathy (DN) is quickly becoming the largest cause of end-stage renal disease (ESRD) in diabetic patients, as well as a major source of morbidity and mortality. Our previous studies indicated that the activation of Nrf2/ARE pathway via Connexin43 (Cx43) considerably contribute to the prevention of oxidative stress in the procession of DN. Fraxin (Fr), the main active glycoside of Fraxinus rhynchophylla Hance, has been demonstrated to possess many potential pharmacological activities. Whereas, whether Fr could alleviate renal fibrosis through regulating Cx43 and consequently facilitating the activation of Nrf2/ARE pathway needs further investigation. The in vitro results showed that: 1) Fr increased the expression of antioxidant enzymes including SOD1 and HO-1 to inhibit high glucose (HG)-induced fibronectin (FN) and inflammatory cell adhesion molecule (ICAM-1) overexpression; 2) Fr exerted antioxidant effect through activating the Nrf2/ARE pathway; 3) Fr significantly up-regulated the expression of Cx43 in HG-induced glomerular mesangial cells (GMCs), while the knock down of Cx43 largely impaired the activation of Nrf2/ARE pathway induced by Fr; 4) Fr promoted the activation of Nrf2/ARE pathway via regulating the interaction between Cx43 and AKT. Moreover, in accordance with the results in vitro, elevated levels of Cx43, phosphorylated-AKT, Nrf2 and downstream antioxidant enzymes related to Nrf2 were observed in the kidneys of Fr-treated group compared with model group. Importantly, Fr significantly improved renal dysfunction pathological changes of renal fibrosis in diabetic db/db mice. Collectively, Fr could increase the Cx43-AKT-Nrf2/ARE pathway activation to postpone the diabetic renal fibrosis and the up-regulation of Cx43 is probably a novel mechanism in this process.

Connexin32 ameliorates epithelial-to-mesenchymal-transition in diabetic renal tubular via inhibiting NOX4.

Renal tubulointerstitial fibrosis (RIF), characterized by epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (TECs), is the main cause of diabetic renal fibrosis. Oxidative stress plays a pivotal role in the development of diabetic RIF. Connexin32 (Cx32), prominently expressed in renal TECs, has emerged as an important player in the regulation of oxidative stress. However, the role of Cx32 in diabetic RIF has not been explored yet. Here, we showed that adenovirus-mediated Cx32 overexpression suppressed EMT to ameliorate RIF and renal function in STZ-induced diabetic mice, while knockout (KO) of Cx32 exacerbated RIF in diabetic mice. Moreover, overexpression of Cx32 inhibited EMT and the production of extra cellular matrix (ECM) in high glucose (HG) induced NRK-52E cells, whereas knockdown of Cx32 showed the opposite effects. Furthermore, we showed that NOX4, the main source of ROS in renal tubular, was down-regulated by Cx32. Mechanistically, Cx32 down-regulated the expression of PKC alpha in a carboxyl-terminal-dependent manner, thereby inhibiting the phosphorylation at Thr147 of p22phox triggered by PKC alpha, which ultimately repressed the formation of the p22phox-NOX4 complex to reduce the protein level of NOX4. Thus, we establish Cx32 as a novel target and confirm the protection mechanism in RIF.

USP9X deubiquitinates connexin43 to prevent high glucose-induced epithelial-to-mesenchymal transition in NRK-52E cells.

Epithelial-to-mesenchymal transition (EMT) plays an important role in diabetic nephropathy (DN). Ubiquitin-specific protease 9X (USP9X/FAM) is closely linked to TGF-ß and fibrosis signaling pathway. However, it remains unknown whether USP9X is involved in the process of EMT in DN. Our previous study has shown that connexin 43 (Cx43) activation attenuated the development of diabetic renal tubulointerstitial fibrosis (RIF). Here, we showed that USP9X is a novel negative regulator of EMT and the potential mechanism is related to the deubiquitination and degradation of Cx43. To explore the potential regulatory mechanism of USP9X, the expression and activity of USP9X were studied by CRISPR/Cas9-based synergistic activation mediator (SAM) system, short hairpin RNAs, and selective inhibitor. The following findings were observed: (1) Expression of USP9X was down-regulated in the kidney tissue of db/db diabetic mice; (2) overexpression of USP9X suppressed high glucose (HG)-induced expressions of EMT markers and extra cellular matrix (ECM) in NRK-52E cells; (3) depletion of USP9X further aggravated EMT process and ECM production in NRK-52E cells; (4) USP9X deubiquitinated Cx43 and suppressed its degradation to regulate EMT process; (5) USP9X deubiquitinated Cx43 by directly binding to the C-terminal Tyr286 of Cx43. The current study determined the protective role of USP9X in the process of EMT and the molecular mechanism clarified that the protective effects of USP9X on DN were associated with the deubiquitination of Cx43.

CKIP-1 acts downstream to Cx43 on the activation of Nrf2 signaling pathway to protect from renal fibrosis in diabetes.

We previously reported that both Cx43 and CKIP-1 attenuated diabetic renal fibrosis via the activation of Nrf2 signaling pathway. However, whether CKIP-1, a scaffold protein, participates in regulating the activation of Nrf2 signaling pathway by Cx43 remains to be elucidated. In this study, the effect of adenovirus-mediated Cx43 overexpression on renal fibrosis in CKIP-1-/- diabetic mice was investigated. We found that overexpression of Cx43 could significantly alleviate renal fibrosis by activating the Nrf2 pathway in diabetic mice, but have no obvious effect in CKIP-1-/- diabetic mice. Cx43 overexpressed plasmid and CKIP-1 small interfering RNA were simultaneously transfected into glomerular mesangial cells and the result demonstrated that the effect of activation of Nrf2 signaling pathway by Cx43 was blocked by CKIP-1 depletion. The interaction between Cx43 and CKIP-1 was analyzed by immunofluorescence and immunoprecipitation assays. We found that Cx43 interacted with CKIP-1, and the interaction was weakened by high glucose treatment. Moreover, Cx43 regulated the expression of CKIP-1 and the interaction of CKIP-1 with Nrf2 via Cx43 carboxyl terminus (CT) domain, thereby activating Nrf2 signaling pathway. According to the results, we preliminary infer that CKIP-1 acts downstream to CX43 on the activation of Nrf2 signaling pathway to protect from renal fibrosis in diabetes, the mechanism of which might be related to the interaction of CKIP-1 with Nrf2 through Cx43 CT. Our study provides further experimental basis for targeting the Cx43-CKIP-1-Nrf2 axis to resist diabetic renal fibrosis.

Polydatin attenuates renal fibrosis in diabetic mice through regulating the Cx32-Nox4 signaling pathway.

We previously found that polydatin could attenuate renal oxidative stress in diabetic mice and improve renal fibrosis. Recent evidence shows that NADPH oxidase 4 (Nox4)-derived reactive oxygen species (ROS) contribute to inflammatory and fibrotic processes in diabetic kidneys. In this study we investigated whether polydatin attenuated renal fibrosis by regulating Nox4 in vitro and in vivo. In high glucose-treated rat glomerular mesangial cells, polydatin significantly decreased the protein levels of Nox4 by promoting its K48-linked polyubiquitination, thus inhibited the production of ROS, and eventually decreasing the expression of fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1), the main factors that exacerbate diabetic renal fibrosis. Overexpression of Nox4 abolished the inhibitory effects of polydatin on FN and ICAM-1 expression. In addition, the expression of Connexin32 (Cx32) was significantly decreased, which was restored by polydatin treatment. Cx32 interacted with Nox4 and reduced its protein levels. Knockdown of Cx32 abolished the inhibitory effects of polydatin on the expression of FN and ICAM-1. In the kidneys of streptozocin-induced diabetic mice, administration of polydatin (100 mg·kg-1·d-1, ig, 6 days a week for 12 weeks) increased Cx32 expression and reduced Nox4 expression, decreased renal oxidative stress levels and the expression of fibrotic factors, eventually attenuating renal injury and fibrosis. In conclusion, polydatin promotes K48-linked polyubiquitination and degradation of Nox4 by restoring Cx32 expression, thereby decreasing renal oxidative stress levels and ultimately ameliorating the pathological progress of diabetic renal fibrosis. Thus, polydatin reduces renal oxidative stress levels and attenuates diabetic renal fibrosis through regulating the Cx32-Nox4 signaling pathway.

Connexin32 ameliorates renal fibrosis in diabetic mice by promoting K48-linked NADPH oxidase 4 polyubiquitination and degradation.

BACKGROUND AND PURPOSE: Nox4 is the major isoform of NADPH oxidase found in the kidney and contributes to the pathogenesis of diabetic nephropathy. However, the molecular mechanisms of increased Nox4 expression induced by hyperglycaemia remain to be elucidated. Here, the role of the connexin32-Nox4 signalling axis in diabetic nephropathy and its related mechanisms were investigated. EXPERIMENTAL APPROACH: Diabetes was induced in mice by low-dose streptozotocin (STZ) combined with a high-fat diet. Effects of connexin32 on Nox4 expression and on renal function and fibrosis in STZ-induced diabetic mice were investigated using adenovirus-overexpressing connexin32 and connexin32-deficient mice. Interactions between connexin32 and Nox4 were analysed by co-immunoprecipitation and immunofluorescence assays. KEY RESULTS: Connexin32 was down-regulated in the kidneys of STZ-induced diabetic mice. Overexpression of connexin32 reduced expression of Nox4 and improved renal function and fibrosis in diabetic mice, whereas connexin32 deficiency had opposite effects. Down-regulation of fibronectin expression by connexin32 was not dependent on gap junctional intercellular communication involving connexin32. Connexin32 interacted with Nox4 and reduced the generation of hydrogen peroxide, leading to the down-regulation of fibronectin expression. Mechanistically, connexin32 decreased Nox4 expression by promoting its K48-linked polyubiquitination. Interestingly, Smurf1 overexpression inhibited K48-linked polyubiquitination of Nox4. Furthermore, connexin32 interacted with Smurf1 and inhibited its expression. CONCLUSION AND IMPLICATIONS: Connexin32 ameliorated renal fibrosis in diabetic mice by promoting K48-linked Nox4 polyubiquitination and degradation via inhibition of Smurf1 expression. Targeting the connexin32-Nox4 signalling axis may contribute to the development of novel treatments for diabetic nephropathy.

Gentiopicroside activates the bile acid receptor Gpbar1 (TGR5) to repress NF-kappaB pathway and ameliorate diabetic nephropathy.

Our previous studies indicated that the G-protein-coupled bile acid receptor, Gpbar1 (TGR5), inhibits inflammation by inhibiting the NF-κB signalling pathway, eventually attenuating diabetic nephropathy (DN). Gentiopicroside (GPS), the main active secoiridoid glycoside of Gentiana manshurica Kitagawa, has been demonstrated to inhibit inflammation in various diseases via inhibiting the inflammatory signalling pathways. However, whether GPS inhibits the NF-κB signalling pathway by activating TGR5 and regulates the pathological progression of diabetic renal fibrosis requires further investigation. In this study, we found that GPS significantly reversed the downregulation of TGR5 and inhibited the overproduction of fibronectin (FN), transforming growth factor ß1 (TGF-ß1), intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) in glomerular mesangial cells (GMCs) exposed to high glucose (HG). Additionally, GPS prevented the phosphorylation and degradation of IκBα, and subsequently inhibited the activation of the NF-κB signalling pathway. Further investigation found that GPS enhanced the stabilization of IκBα by promoting the interaction of ß-arrestin2 with IκBα via TGR5 activation, which contributed to the inhibition of NF-κB signalling pathway. Importantly, the depletion of TGR5 blocked the inhibition of the NF-κB signalling pathway and reversed the downregulation of FN, ICAM-1, VCAM-1 and TGF-ß1 by GPS in HG-induced GMCs. Moreover, GPS increased the TGR5 protein levels and promoted the interaction between IκBα and ß-arrestin2, thereby inhibiting the reduction of IκBα and blocked NF-κB p65 nuclear translocation in the kidneys of STZ-induced diabetic mice. Collectively, these data suggested that GPS regulates the TGR5-ß-arrestin2-NF-κB signalling pathway to prevent inflammation in the kidneys of diabetic mice, and ultimately ameliorates the pathological progression of diabetic renal fibrosis.

PAQR3 regulates phosphorylation of FoxO1 in insulin-resistant HepG2 cells via NF-κB signaling pathway.

Insulin resistance is a significant feature of type 2 diabetes mellitus and glucose and lipid metabolism disorders. Activation of NF-κB signaling pathway plays an important role in the formation of insulin resistance. FoxO1 plays a major role in regulating glucose and lipid metabolism, as well as insulin signaling pathway. Previous studies have shown that Progestin and AdipoQ Receptor 3 (PAQR3) suppresses the activity of PI3K/Akt, which is an upstream pathway of FoxO1, and additionally promotes the pathological process of diabetic renal inflammatory fibrosis via activating NF-κB pathway. On this basis, it has caused us great concern whether NF-κB is involved in PAQR3 regulation of FoxO1 under insulin resistance. In this study, we aimed to investigate whether PAQR3 regulates phosphorylation of FoxO1 via NF-κB pathway in palmitic acid (PA)-induced insulin-resistant HepG2 cells, thereby causing glucose and lipid metabolism disorders. We found that PA stimulation and PAQR3 overexpression decreased the phosphorylation of FoxO1 and the expressions of glucokinase (GCK) and low density lipoprotein receptor (LDLR), in addition, promoted the nuclear accumulation of NF-κB. Inhibition of NF-κB pathway increased the phosphorylation of FoxO1 and the expressions of GCK and LDLR which were downregulated by PA stimulation and PAQR3 overexpression. Taken together, in PA-induced insulin-resistant HepG2 cells, PAQR3 might regulate the phosphorylation of FoxO1 and the expressions of GCK and LDLR through NF-κB pathway, thereby regulating the glucose and lipid metabolism disorders induced by insulin resistance.

Paeonol Ameliorates Glucose and Lipid Metabolism in Experimental Diabetes by Activating Akt.

Our previous study proved that paeonol (Pae) could lower blood glucose levels of diabetic mice. There are also a few reports of its potential use for diabetes treatment. However, the role of Pae in regulating glucose and lipid metabolism in diabetes remains largely unknown. Considering the critical role of serine/threonine kinase B (Akt) in glucose and lipid metabolism, we explored whether Pae could improve glucose and lipid metabolism disorders via Akt. Here, we found that Pae attenuated fasting blood glucose, glycosylated serum protein, serum cholesterol and triglyceride (TG), hepatic glycogen, cholesterol and TG in diabetic mice. Moreover, Pae enhanced glucokinase (GCK) and low-density lipoprotein receptor (LDLR) protein expressions, and increased the phosphorylation of Akt. In insulin-resistant HepG2 cells, Pae increased glucose uptake and decreased lipid accumulation. What's more, Pae elevated LDLR and GCK expressions as well as Akt phosphorylation, which was consistent with the in vivo results. Knockdown and inhibition experiments of Akt revealed that Pae regulated LDLR and GCK expressions through activation of Akt. Finally, molecular docking assay indicated the steady hydrogen bond was formed between Pae and Akt2. Experiments above suggested that Pae ameliorated glucose and lipid metabolism disorders and the underlying mechanism was closely related to the activation of Akt.