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Diabetic kidney disease (DKD) is a major microvascular complication of diabetes mellitus (DM) and is closely associated to programmed cell death. However, the complex mechanisms of necroptosis, an alternative cell death pathway, in DKD pathogenesis are yet to be elucidated. This study indicates that necroptosis is involved in DKD induced by high glucose (HG) both in vivo and in vitro. HG intervention led to the activation of RIPK1/RIPK3/MLKL signaling, resulting in renal tissue necroptosis and proinflammatory activation in streptozotocin/high-fat diet- (STZ/HFD-) induced diabetic mice and HG-induced normal rat kidney tubular cells (NRK-52E). We further found that in HG-induced NRK-52E cell, necroptosis might, at least partly, depend on the levels of reactive oxygen species (ROS). Meanwhile, ROS participated in necroptosis via a positive feedback loop involving the RIPK1/RIPK3 pathway. In addition, blocking RIPK1/RIPK3/MLKL signaling by necrostatin-1 (Nec-1), a key inhibitor of RIPK1 in the necroptosis pathway, or antioxidant N-acetylcysteine (NAC), an inhibitor of ROS generation, could effectively protect the kidney against HG-induced damage, decrease the release of proinflammatory cytokines, and rescue renal function in STZ/HFD-induced diabetic mice. Inhibition of RIPK1 effectively decreased the activation of RIPK1-kinase-/NF- κ B-dependent inflammation. Collectively, we demonstrated that high glucose induced DKD via renal tubular epithelium necroptosis, and Nec-1 or NAC treatment downregulated the RIPK1/RIPK3/MLKL pathway and finally reduced necroptosis, oxidative stress, and inflammation. Thus, RIPK1 may be a therapeutic target for DKD., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Man Guo et al.)

Diabetic nephropathy (DN) is a chronic low-grade inflammatory disease. Oxidative stress and nuclear factor kappa B (NF- κ B) signaling play an important role in the pathogenesis of DN. Short-chain fatty acids (SCFAs) produced from carbohydrate fermentation in the gastrointestinal tract exert positive regulatory effects on inflammation and kidney injuries. However, it is unclear whether SCFAs can prevent and ameliorate DN. In the present study, we evaluated the role and mechanism of the three main SCFAs (acetate, propionate, and butyrate) in high-fat diet (HFD) and streptozotocin- (STZ-) induced type2 diabetes (T2D) and DN mouse models and in high glucose-induced mouse glomerular mesangial cells (GMCs), to explore novel therapeutic strategies and molecular targets for DN. We found that exogenous SCFAs, especially butyrate, improved hyperglycemia and insulin resistance; prevented the formation of proteinuria and an increase in serum creatinine, urea nitrogen, and cystatin C; inhibited mesangial matrix accumulation and renal fibrosis; and blocked NF- κ B activation in mice. SCFAs also inhibited high glucose-induced oxidative stress and NF- κ B activation and enhanced the interaction between β -arrestin-2 and I- κ B α in GMCs. Specifically, the beneficial effects of SCFAs were significantly facilitated by the overexpression GPR43 or imitated by a GPR43 agonist but were inhibited by siRNA-GPR43 in GMCs. These results support the conclusion that SCFAs, especially butyrate, partially improve T2D-induced kidney injury via GPR43-mediated inhibition of oxidative stress and NF- κ B signaling, suggesting SCFAs may be potential therapeutic agents in the prevention and treatment of DN., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2020 Wei Huang et al.)

Aim: To explore the role of group 2 innate lymphoid cells (ILC2s) in the pathogenesis of renal fibrosis in diabetic kidney disease (DKD)., Methods: The proportion of ILC2s and the levels of Th2 cytokines (IL-4, IL-5, and IL-13) in the peripheral blood of normal control subjects (NC) or patients with type 2 diabetes mellitus (DM), early diabetic kidney disease (DKD1), or late diabetic kidney disease (DKD2) were analyzed by flow cytometry and ELISA. The expression of transforming growth factor- β 1 (TGF- β 1), fibronectin (FN), collagen1, IL-4R α , and IL-13R α 1 in renal tubular epithelial cells (HK-2) induced by IL-4, IL-13, or high glucose was analyzed by ELISA or qPCR., Results: The proportion of ILC2s and the levels of IL-4, IL-5, and IL-13 were significantly increased in DKD patients and were positively correlated with the severity of DKD ( P < 0.05). The expression of TGF- β 1, FN, and collagen1 was significantly upregulated in HK-2 cells induced by IL-4 or IL-13 ( P < 0.05). Moreover, the IL-4R α and IL-13R α 1 mRNA in HK2 cells were increased followed by high glucose alone or combined with IL-4 or IL-13, but the differences were not statistically significant ( P > 0.05). However, compared with high-glucose stimulation alone, the expression of TGF- β 1, FN, and collagen1 was significantly increased in HK-2 cells induced by high glucose combined with IL-4 or IL-13 ( P < 0.05)., Conclusions: ILC2s may participate in renal fibrosis in DKD partly via TGF- β 1 signal pathway.

Aims: Abnormal regulation of autophagy participates in the development of diabetic nephropathy. mTOR is the most common negative regulator of the autophagy signaling pathway. FBW7 constitutes the SCF (Skp1-Cullin1-F-box protein) recognition subunit of E3 ubiquitin ligase, and mTOR is a substrate of FBW7 that can be modified by ubiquitination and be degraded via proteasomes. In this study, we explored the relationship between FBW7 and autophagy and examined the effects of FBW7 on the occurrence of diabetic nephropathy in vitro., Materials and Methods: We cultured mesangial cells induced by high glucose in vitro and used rapamycin as a specific mTOR inhibitor, performed FBW7 gene overexpression, and detected the expression of autophagy signal and inflammatory factors by WB, ELISA, RT-PCR, and immunofluorescence., Results: High glucose can downregulate the expression of FBW7 and activate mTOR signal, which leads to diminished autophagy in renal mesangial cells, as well as renal inflammatory cytokines and fibrotic factors. RAPA, as a specifically inhibitor of mTOR, can decrease inflammatory cytokines and fibrotic factors by inhibiting mTOR. Moreover, FBW7 gene overexpression can increase autophagy by inhibiting mTOR signal; at the same time, the inflammatory cytokines and fibrotic factors were decreased in mesangial cells., Conclusions: FBW7 was decreased in renal mesangial cells induced by high glucose, and FBW7 gene overexpression can increase autophagy by inhibiting mTOR signaling and ameliorate inflammation and fibrosis.

Previous studies demonstrated that ROS-NLRP3 inflammasome signaling activation was involved in the pathogenesis of diabetic nephropathy (DN). Recent research has shown that sweet taste receptors (STRs) are important sentinels of innate immunity. Whether high glucose primes ROS-NLRP3 inflammasome signaling via STRs is unclear. In this study, diabetic mouse model was induced by streptozotocin (STZ) in vivo; mouse glomerular mesangial cells (GMCs) and human proximal tubular cells were stimulated by high glucose (10, 20, and 30 mmol/L) in vitro; STR inhibitor lactisole was used as an intervention reagent to evaluate the role and mechanism of the STRs in the pathogenesis of DN. Our results showed that the expression of STRs and associated signaling components (G α -gustducin, PLC β 2, and TRPM5) was obviously downregulated under the condition of diabetes in vivo and in vitro. Furthermore, lactisole significantly mitigated the production of intracellular ROS and reversed the high glucose-induced decrease of Ca 2+ and the activation of NLRP3 inflammasome signaling in vitro ( p < 0.05). These combined results support the hypothesis that STRs could be involved in the activation of ROS-NLRP3 inflammasome signaling in the pathogenesis of DN, suggesting that STRs may act as new therapeutic targets of DN.

Nuclear factor-kappa B (NF- κ B) is the key part of multiple signal transduction of inflammation in the pathogenesis of diabetic nephropathy (DN). The ubiquitin-proteasome system is extensively involved in the regulation of the NF- κ B pathway. Cylindromatosis (CYLD) has deubiquitinase activity and acts as a negative regulator of the NF- κ B signaling pathway. However, the association between CYLD and NF- κ B inflammatory signaling in DN is unclear. In the present study, mouse glomerular mesangial cells (GMCs) and rat GMCs were stimulated by elevated concentrations of glucose (10, 20, and 30 mmol/L high glucose) or mannitol as the osmotic pressure control. CYLD was overexpressed or suppressed by transfection with a CYLD expressing vector or CYLD-specific siRNA, respectively. Our data showed that high glucose significantly inhibited the protein and mRNA expression of CYLD in a dose- and time-dependent manner (both p < 0.05). siRNA-mediated knockdown CYLD facilitated the high glucose-induced activation of NF- κ B signaling and triggered the release of MCP-1, IL-6, and IL-8 (all p < 0.05). However, these high glucose-mediated effects were blunted by overexpression of CYLD ( p < 0.05). The present results support the involvement of CYLD in the regulation of NF- κ B inflammatory signaling induced by elevated glucose, implicating CYLD as a potential therapeutic target of DN.

Background: Diabetic nephropathy is a common complication of diabetes mellitus. This study aimed to explore whether mesenchymal stem cells (MSCs) transplantation could attenuate diabetic nephropathy in experimental diabetic rats., Methods: Sprague-Dawley rats received a single intraperitoneal injection of streptozotocin (STZ) (60 mg/kg). Diabetic rats were randomized to four groups: diabetes control group (DC), ciclosporin A group (CsA), MSC group, and MSC + CsA group (MSCA). Bone marrow mesenchymal stem cells were cultured, identified and labeled by 5-bromo-2'-deoxyuridine (BrdU) in vitro. Then they were transplanted to diabetic rats via introcardiac infusion. Ciclosporin A was administered daily at 5 mg/kg. At 1, 2, 4, 8 weeks after transplantation, random blood glucose, urine albumin/creatinine ratio (Alb/Cr), endogenous creatinine clearance rate and renal mass index were tested. Renal morphology and labeled cells were examined., Results: Cultured MSCs expressed mesenchymal cell phenotype, and could be multidifferentiated to osteogenic and adipogenic cells. Labeled MSCs could be detected in the kidney of nephropathic rats, mainly in renal interstitium, but they did not propagate after engrafting in kidney. Over the course of the experiment, MSCA group showed a significant decrease in blood glucose compared with MSC group, CsA group and DC group (P < 0.05, respectively). The Alb/Cr in MSCA group and MSC group were significantly lower than CsA group and DC group (P < 0.05). And the Alb/Cr in MSCA group showed a significant decrease compared with MSC group (0.74 vs 0.84, P < 0.05). There was a significant difference in renal mass index between the MSCA group and DC group (5.66 vs 6.37, P < 0.05). No significant difference was found in creatinine clearance rate among 4 groups (P > 0.05). Treatment with MSC + CsA significantly ameliorated the morphology of diabetic kidney., Conclusion: MSC could mildly ameliorate diabetic nephropathy by decreasing blood glucose, Alb/Cr ratio and renal mass index.

Purpose: To establish nomograms integrating serum lactate levels and traditional risk factors for predicting diabetic kidney disease (DKD) in type 2 diabetes mellitus (T2DM) patients. Patients and methods: A total of 570 T2DM patients and 100 healthy subjects were enrolled. T2DM patients were categorized into normal and high lactate groups. Univariate and multivariate logistic regression analyses were employed to identify independent predictors for DKD. Then, nomograms for predicting DKD were established, and the model performance was evaluated using the area under the receiver operating characteristic curve (AUC), calibration, and decision curve analysis (DCA). Results: T2DM patients exhibited higher lactate levels compared to those in healthy subjects. Glucose, platelet, uric acid, creatinine, and hypertension were independent factors for DKD in T2DM patients with normal lactate levels, while diabetes duration, creatinine, total cholesterol, and hypertension were indicators in high lactate levels group (P< 0.05). The AUC values were 0.834 (95% CI, 0.776 to 0.891) and 0.741 (95% CI, 0.688 to 0.795) for nomograms in both normal lactate and high lactate groups, respectively. The calibration curve demonstrated excellent agreement of fit. Furthermore, the DCA revealed that the threshold probability and highest Net Yield were 17– 99% and 0.36, and 24– 99% and 0.24 for the models in normal lactate and high lactate groups, respectively. Conclusion: The serum lactate level-based nomogram models, combined with traditional risk factors, offer an effective tool for predicting DKD probability in T2DM patients. This approach holds promise for early risk assessment and tailored intervention strategies. [ABSTRACT FROM AUTHOR]

Background. Hyperglycemia plays a vital role in diabetic nephropathy (DN); autophagy and its potential upregulator receptor-interacting protein kinase 2 (RIPK2) are associated with ROS, which play a potential role in regulating NLRP3, and may be involved in inflammation in DN. Aim. In this study, we aimed to explore the mechanisms mediated by RIPK2 in autophagy and the relationship with ROS-NLRP3 of DN, by investigating the levels of RIPK2 and autophagy in glomerular mesangial cells (GMCs) stimulated with high glucose. Material and Methods. GMCs were divided into the following groups: normal group (NC), high glucose group (HG), and RIPK2 siRNA group. RIPK2, LC3, caspase1, and IL-1β levels were measured by western blotting and RT-PCR. Autophagosomes were measured by GFP-RFP-LC3; ROS were detected by DCFH-DA. Results. High glucose upregulated RIPK2 and LC3 in GMCs during short periods (0-12 h) (p<0.01), while RIPK2 and LC3 were significantly downregulated in the long term (12-72 h) (p<0.01); these changes were positively correlated with glucose concentration (p<0.01). In addition, levels of ROS, caspase1, and IL-1β increased in a time- and dose-dependent manner in the high glucose group, even with an increased expression of LC3 (p<0.01). However, LC3 expression decreased in the siRIPK2 group, while levels of ROS, caspase1, and IL-1β increased (p<0.01). Conclusions. Autophagy was activated by high glucose at short time periods but was inhibited in the long term, demonstrating a dual role for high glucose in autophagy of GMCs. RIPK2 regulates ROS-NLRP3 inflammasome signaling through autophagy and may be involved in the pathogenesis of DN. [ABSTRACT FROM AUTHOR]

Aim. To explore the role of group 2 innate lymphoid cells (ILC2s) in the pathogenesis of renal fibrosis in diabetic kidney disease (DKD). Methods. The proportion of ILC2s and the levels of Th2 cytokines (IL-4, IL-5, and IL-13) in the peripheral blood of normal control subjects (NC) or patients with type 2 diabetes mellitus (DM), early diabetic kidney disease (DKD1), or late diabetic kidney disease (DKD2) were analyzed by flow cytometry and ELISA. The expression of transforming growth factor-β1 (TGF-β1), fibronectin (FN), collagen1, IL-4Rα, and IL-13Rα1 in renal tubular epithelial cells (HK-2) induced by IL-4, IL-13, or high glucose was analyzed by ELISA or qPCR. Results. The proportion of ILC2s and the levels of IL-4, IL-5, and IL-13 were significantly increased in DKD patients and were positively correlated with the severity of DKD (P<0.05). The expression of TGF-β1, FN, and collagen1 was significantly upregulated in HK-2 cells induced by IL-4 or IL-13 (P<0.05). Moreover, the IL-4Rα and IL-13Rα1 mRNA in HK2 cells were increased followed by high glucose alone or combined with IL-4 or IL-13, but the differences were not statistically significant (P>0.05). However, compared with high-glucose stimulation alone, the expression of TGF-β1, FN, and collagen1 was significantly increased in HK-2 cells induced by high glucose combined with IL-4 or IL-13 (P<0.05). Conclusions. ILC2s may participate in renal fibrosis in DKD partly via TGF-β1 signal pathway. [ABSTRACT FROM AUTHOR]

Nuclear factor-kappa B (NF-κB) is the key part of multiple signal transduction of inflammation in the pathogenesis of diabetic nephropathy (DN). The ubiquitin-proteasome system is extensively involved in the regulation of the NF-κB pathway. Cylindromatosis (CYLD) has deubiquitinase activity and acts as a negative regulator of the NF-κB signaling pathway. However, the association between CYLD and NF-κB inflammatory signaling in DN is unclear. In the present study, mouse glomerular mesangial cells (GMCs) and rat GMCs were stimulated by elevated concentrations of glucose (10, 20, and 30 mmol/L high glucose) or mannitol as the osmotic pressure control. CYLD was overexpressed or suppressed by transfection with a CYLD expressing vector or CYLD-specific siRNA, respectively. Our data showed that high glucose significantly inhibited the protein and mRNA expression of CYLD in a dose- and time-dependent manner (both p<0.05). siRNA-mediated knockdown CYLD facilitated the high glucose-induced activation of NF-κB signaling and triggered the release of MCP-1, IL-6, and IL-8 (all p<0.05). However, these high glucose-mediated effects were blunted by overexpression of CYLD (p<0.05). The present results support the involvement of CYLD in the regulation of NF-κB inflammatory signaling induced by elevated glucose, implicating CYLD as a potential therapeutic target of DN. [ABSTRACT FROM AUTHOR]

Background. Inflammation and fibrosis are the important pathophysiologic processes in diabetic nephropathy (DN). Maresin 1 is a potential anti-inflammatory lipid mediator, which has displayed powerful proresolving activities. Aim. We determine whether maresin 1 has protective effect on mouse glomerular mesangial cells (GMCs) induced by high glucose. Methods. We cultured GMCs stimulated by high glucose and categorized as follows: normal glucose group (5.6 mmol/L), high glucose group (30 mmol/L), mannitol group, maresin 1 intervention group (1, 10, and 100 nmol/L), maresin 1 and normal glucose group, and the N-acetylcysteine (NAC) intervention group (10 μmol/L NAC). After 24 h, the expression of ROS, NLRP3, caspase-1, procaspase-1, IL-1β, and pro-IL-1β was detected by western-blot, RT-PCR, and immunofluorescence. After 48 h, the expression of TGF-β1 and FN was detected by RT-PCR and ELISA. Results. Compared with normal glucose group, the expression of ROS, NLRP3, caspase-1, IL-1β, TGF-β1, and FN increased in high glucose group (P<0.05), but it decreased after the treatment of maresin 1 in different concentrations. On the contrary, the expression of procaspase-1 and pro-IL-1β protein was restrained by high glucose and enhanced by maresin 1 in a dose-dependent manner (P<0.05). Conclusion. Maresin 1 can inhibit NLRP3 inflammasome, TGF-β1, and FN in GMCs; it may have protective effect on DN by mitigating the inflammation and early fibrosis. [ABSTRACT FROM AUTHOR]

While inflammation is considered a central component in the development in diabetic nephropathy, the mechanism remains unclear. The NLRP3 inflammasome acts as both a sensor and a regulator of the inflammatory response. The NLRP3 inflammasome responds to exogenous and endogenous danger signals, resulting in cleavage of procaspase-1 and activation of cytokines IL-1β, IL-18, and IL-33, ultimately triggering an inflammatory cascade reaction. This study observed the expression of NLRP3 inflammasome signaling stimulated by high glucose, lipopolysaccharide, and reactive oxygen species (ROS) inhibitor N-acetyl-L-cysteine in glomerular mesangial cells, aiming to elucidate the mechanism by which the NLRP3 inflammasome signaling pathway may contribute to diabetic nephropathy. We found that the expression of thioredoxin-interacting protein (TXNIP), NLRP3, and IL-1β was observed by immunohistochemistry in vivo. Simultaneously, the mRNA and protein levels of TXNIP, NLRP3, procaspase-1, and IL-1β were significantly induced by high glucose concentration and lipopolysaccharide in a dose-dependent and time-dependent manner in vitro. This induction by both high glucose and lipopolysaccharide was significantly inhibited by N-acetyl-L-cysteine. Our results firstly reveal that high glucose and lipopolysaccharide activate ROS/TXNIP/ NLRP3/IL-1β inflammasome signaling in glomerular mesangial cells, suggesting a mechanism by which inflammation may contribute to the development of diabetic nephropathy. [ABSTRACT FROM AUTHOR]