Paeoniflorin protects against cisplatin-induced acute kidney injury through targeting Hsp90AA1-Akt protein-protein interaction.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeonia lactiflora Pall has been used in Chinese Medicine for thousands of years, especially having anti-inflammatory, sedative, analgesic and other ethnic pharmacological effects. Moreover, Paeoniflorin is the main active ingredient of the Paeonia lactiflora Pall, and most are used in the treatment of inflammation-related autoimmune diseases. In recent years, studies have found that Paeoniflorin has a therapeutic effect on a variety of kidney diseases. AIM OF THE STUDY: Cisplatin (CIS) is limited in clinical use due to its serious side effects, such as renal toxicity, and there is no effective method for prevention. Paeoniflorin (Pae) is a natural polyphenol which has a protective effect against many kidney diseases. Therefore, our study is to explore the effect of Pae on CIS-induced AKI and the specific mechanism. MATERIALS AND METHODS: Firstly, CIS induced acute renal injury model was constructed in vivo and in vitro, and Pae was continuously injected intraperitoneally three days in advance, and then Cr, BUN and renal tissue PAS staining were detected to comprehensively evaluate the protective effect of Pae on CIS-induced AKI. We then combined Network Pharmacology with RNA-seq to investigate potential targets and signaling pathways. Finally, affinity between Pae and core targets was detected by molecular docking, CESTA and SPR, and related indicators were detected in vitro and in vivo. RESULTS: In this study, we first found that Pae significantly alleviated CIS-AKI in vivo and in vitro. Through network pharmacological analysis, molecular docking, CESTA and SPR experiments, we found that the target of Pae was Heat Shock Protein 90 Alpha Family Class A Member 1 (Hsp90AA1) which performs a crucial function in the stability of many client proteins including Akt. RNA-seq found that the KEGG enriched pathway was PI3K-Akt pathway with the most associated with the protective effect of Pae which is consistent with Network Pharmacology. GO analysis showed that the main biological processes of Pae against CIS-AKI include cellular regulation of inflammation and apoptosis. Immunoprecipitation further showed that pretreatment with Pae promoted the Hsp90AA1-Akt protein-protein Interactions (PPIs). Thereby, Pae accelerates the Hsp90AA1-Akt complex formation and leads to a significant activate in Akt, which in turn reduces apoptosis and inflammation. In addition, when Hsp90AA1 was knocked down, the protective effect of Pae did not continue. CONCLUSION: In summary, our study suggests that Pae attenuates cell apoptosis and inflammation in CIS-AKI by promoting Hsp90AA1-Akt PPIs. These data provide a scientific basis for the clinical search for drugs to prevent CIS-AKI.

Paeoniflorin binds to VEGFR2 to restore autophagy and inhibit apoptosis for podocyte protection in diabetic kidney disease through PI3K-AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin (PF) was found to exhibit renal protection from diabetic kidney disease (DKD) in previous trials, but its specific mechanism remains to be elucidated. AIM OF THE STUDY: This study furtherly explored the specific mechanism of PF in protect podocyte injury in DKD. MATERIALS AND METHODS: We observed the effects of PF on renal tissue and podocytes in DKD by constructing the vitro and vivo models after measuring the pharmacokinetic characteristics of PF. Target proteins of PF were found through target prediction, and verified by molecular docking, CESTA, and SPR, and then furtherly explored the downstream regulation mechanism related to podocyte autophagy and apoptosis by network prediction and co-immunoprecipitation. Finally, by using the target protein inhibitor in vivo and knocking down the target protein gene in vitro, it was verified that PF played a role in regulating autophagy and apoptosis through the target protein in diabetic nephropathy. RESULTS: This study found that in STZ-induced mice model, PF could improve the renal biochemical and pathological damage and podocyte injure (p < 0.05), upregulate autophagy activity (p < 0.05), but inhibit apoptosis (p < 0.01). Vascular endothelial growth factor receptor 2 (VEGFR2), predicted as the target of PF, directly bind with PF reflected by molecular docking and surface plasmon resonance detection. Animal studies demonstrated that VEGFR2 inhibitors have a protective effect similar to that of PF on DKD. Network prediction and co-immunoprecipitation further confirmed that VEGFR2 was able to bind PIK3CA to regulate PI3K-AKT signaling pathway. Furthermore, PF downregulated the phosphorylation of PI3K and AKT (p < 0.05). In vitro, similarly to autophagy inhibitors, PF was also found to improve podocyte markers (p < 0.05) and autophagy activity (p < 0.05), decrease caspase 3 protein (p < 0.05) and further inhibited VEGFR2-PI3K-AKT activity (p < 0.05). Finally, the results of VEGFR2 knockdown were similar to the effect of PF in HG-stimulated podocytes. CONCLUSION: In conclusion, PF restores autophagy and inhibits apoptosis by targeting the VEGFR2-mediated PI3K-AKT pathway to improve renal injury in DKD, that provided a theoretical basis for PF treatment in DKD.

Paeoniflorin directly binds to TNFR1 to regulate podocyte necroptosis in diabetic kidney disease.

Necroptosis was elevated in both tubulointerstitial and glomerular renal tissue in patients with diabetic kidney disease (DKD), and was most pronounced on glomerulus in the stage with macroalbuminuria. This study further explored whether paeoniflorin (PF) could affect podocyte necroptosis to protect kidney injure in vivo and in vitro. Our study firstly verified that there are obvious necroptosis-related changes in the glomeruli of DKD through bioinformatics analysis combined with clinicopathological data. STZ-induced mouse diabetes model and high-glucose induced podocyte injury model were used to evaluate the renoprotection, podocyte injury protection and necroptosis regulation of PF in DKD. Subsequently, the target protein-TNFR1 that PF acted on podocytes was found by computer target prediction, and then molecular docking and Surface plasmon resonance (SPR) experiments were performed to verify that PF had the ability to directly bind to TNFR1 protein. Finally, knockdown of TNFR1 on podocytes in vitro verified that PF mainly regulated the programmed necrosis of podocytes induced by high glucose through TNFR1. In conclusion, PF can directly bind and promote the degradation of TNFR1 in podocytes and then regulate the RIPK1/RIPK3 signaling pathway to affect necroptosis, thus preventing podocyte injury in DKD. Thus, TNFR1 may be used as a new potential target to treat DKD.

Exosomal miR-7002-5p derived from highglucose-induced macrophages suppresses autophagy in tubular epithelial cells by targeting Atg9b.

Macrophage infiltration plays an important role in the progression of diabetic nephropathy (DN). Previously, we demonstrated that highglucose-stimulated macrophage-derived exosomes (HG-exo) induces proliferation and extracellular matrix accumulation in glomerular mesangial cells, but its effect on tubular cells is unclear. This study aimed to explore the role of HG-exo on renal tubular injury in DN. The results show that HG-exo could induce dysfunction, autophagy inhibition, and inflammation in mouse tubular epithelial cell (mTEC) and C57 mouse kidney. Moreover, miR-7002-5p was differentially expressed in HG-exo based on miRNAs sequencing and bioinformatics analysis. A dual-luciferase reporter assay confirmed that Atg9b was the direct target gene of miR-7002-5p. Further experimentation showed that miR-7002-5p inhibition in vivo and vitro reserves HG-exo effects. These results demonstrated that HG-exo carries excessive miR-7002-5p and inhibits autophagy through targeting Atg9b; this process then induces renal tubular dysfunction and inflammation. In conclusion, our study clarifies the important role of macrophage-derived exosomes in DN and is expected to provide new insight on DN prevention and treatment.

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.

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-ß-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.

Wogonin protects glomerular podocytes by targeting Bcl-2-mediated autophagy and apoptosis in diabetic kidney disease.

Diabetic kidney disease (DKD) is one of the microvascular complications of diabetes mellitus and a major cause of end-stage renal disease with limited treatment options. Wogonin is a flavonoid derived from the root of Scutellaria baicalensis Georgi, which has shown a potent renoprotective effect. But the mechanisms of action in DKD are not fully elucidated. In this study, we investigated the effects of wogonin on glomerular podocytes in DKD using mouse podocyte clone 5 (MPC5) cells and diabetic mice model. MPC5 cells were treated with high glucose (30 mM). We showed that wogonin (4, 8, 16 µM) dose-dependently alleviated high glucose (HG)-induced MPC5 cell damage, accompanied by increased expression of WT-1, nephrin, and podocin proteins, and decreased expression of TNF-α, MCP-1, IL-1ß as well as phosphorylated p65. Furthermore, wogonin treatment significantly inhibited HG-induced apoptosis in MPC5 cells. Wogonin reversed HG-suppressed autophagy in MPC5 cells, evidenced by increased ATG7, LC3-II, and Beclin-1 protein, and decreased p62 protein. We demonstrated that wogonin directly bound to Bcl-2 in MPC5 cells. In HG-treated MPC5 cells, knockdown of Bcl-2 abolished the beneficial effects of wogonin, whereas overexpression of Bcl-2 mimicked the protective effects of wogonin. Interestingly, we found that the expression of Bcl-2 was significantly decreased in biopsy renal tissue of diabetic nephropathy patients. In vivo experiments were conducted in STZ-induced diabetic mice, which were administered wogonin (10, 20, 40 mg · kg-1 · d-1, i.g.) every other day for 12 weeks. We showed that wogonin administration significantly alleviated albuminuria, histopathological lesions, and p65 NF-κB-mediated renal inflammatory response. Wogonin administration dose-dependently inhibited podocyte apoptosis and promoted podocyte autophagy in STZ-induced diabetic mice. This study for the first time demonstrates a novel action of wogonin in mitigating glomerulopathy and podocytes injury by regulating Bcl-2-mediated crosstalk between autophagy and apoptosis. Wogonin may be a potential therapeutic drug against DKD.

Carnosine alleviates podocyte injury in diabetic nephropathy by targeting caspase-1-mediated pyroptosis.

Diabetic nephropathy (DN) is a main complication of diabetes and often develops into end-stage nephropathy. Histologically, DN progresses as the gradual loss of podocytes with the loss of glomerular podocytes being the earliest sign of DN. Pyroptosis is a new type of programmed cell death and has been mechanistically correlated with podocyte injury in DN. The current study aimed to evaluate the protective effects of carnosine on glomerular podocytes in DN, both in vivo and in vitro. Using high glucose-treated cultured MPC5 cells and a streptozotocin (STZ)-induced diabetic mouse model, we evaluated the effects of carnosine on alleviating podocyte injury in DN. We found that carnosine significantly reversed albuminuria and histopathological lesions and alleviated renal inflammatory and pyroptosis responses in STZ-induced diabetic mice for 12 weeks. The results also showed that carnosine strongly inhibited podocyte inflammation and podocyte pyroptosis in vitro. Cellular Thermal Shift Assay (CETSA) and molecular docking results revealed that mechnaistically caspase-1 was the target of carnosine. We then found that silencing caspase-1 eliminated the protective effect of carnosine. Interestingly, we also found that caspase-1 and gasdermin D expression were increased in renal biopsy tissue of patients with DN. Our study is the first to demonstrate the novel role of carnosine in alleviating podocyte injury by inhibiting pyroptosis via the targeting of caspase-1. Carnosine may have potential as a therapeutic agent in treating DN by targeting caspase-1.

Complement-mediated M2/M1 macrophage polarization may be involved in crescent formation in lupus nephritis.

The function of the complement and macrophage crosstalk during the formation of crescents in lupus nephritis has not yet been reported. This study therefore aimed to explore the association of crescents, complements, and M2 macrophages with clinical features in lupus nephritis. We assessed a Chinese cohort comprising 301 patients with lupus nephritis. Renal biopsy specimens were collected from 64 patients with proliferative lupus nephritis (class III/III + V or IV/IV + V). The renal deposition of cluster of differentiation (CD) 68, inducible nitric oxide synthase, CD163, and C3a receptor (C3aR) was evaluated by immunostaining. The associations among crescents, complements, and M2 macrophages were also analyzed. Next, the underlying mechanism was investigated in vitro using C3a-treated macrophages. We found that M2-phenotype macrophages (CD163+) were the dominant subpopulation in human lupus nephritis. Additionally, a significant association was observed among the CD163+ macrophages, crescents, and complement activation. C3aR co-localized with CD163 and correlated with crescents and could induce polarization of macrophages to an M2 phenotype. Overall, these results suggest that complement-mediated M2/M1 macrophage polarization may contribute to the formation of crescents in lupus nephritis.

Bruton's tyrosine kinase regulates macrophage­induced inflammation in the diabetic kidney via NLRP3 inflammasome activation.

It has been previously reported that macrophages may be involved in diabetic nephropathy (DN) development. Furthermore, Bruton's tyrosine kinase (BTK) may participate in macrophage activation and lead to the release of inflammatory mediators. The main aim of the present study was to analyze the association between renal BTK expression and clinical indicators. Moreover, BTK knockout mice were used to establish a diabetic model for further research. The results demonstrated that BTK was activated in the kidneys of patients with DN and was associated with the progression of proteinuria, creatinine levels, estimated glomerular filtration rate and pathological changes in the kidneys of patients with DN. Furthermore, BTK knockout was observed to reduce urinary protein excretion, alleviate renal injury and decrease renal inflammation in diabetic mice. This protection may be attributed to BTK­induced suppression of the activation of the Nod­like receptor (NLR) family pyrin domain containing 3 inflammasome. Collectively, it has been demonstrated in the present study that BTK may be a potential target for DN treatment.

Wogonin Alleviates Kidney Tubular Epithelial Injury in Diabetic Nephropathy by Inhibiting PI3K/Akt/NF-κB Signaling Pathways.

INTRODUCTION: Kidney tubular epithelial injury is one of the key factors in the progression of diabetic nephropathy (DN). Wogonin is a kind of flavonoid, which has many pharmacological effects, such as anti-inflammation, anti-oxidation and anti-fibrosis. However, the effect of wogonin in renal tubular epithelial cells during DN is still unknown. MATERIALS AND METHODS: STZ-induced diabetic mice were given doses of wogonin (10, 20, and 40 mg/kg) by intragastric administration for 16 weeks. The metabolic indexes from blood and urine and pathological damage of renal tubules in mice were evaluated. Human tubular epithelial cells (HK-2) were cultured in high glucose (HG) condition containing wogonin (2µM, 4µM, 8µM) for 24 h. Tubular epithelial cell inflammation and autophagic dysfunction both in vivo and in vitro were assessed by Western blot, qRT-PCR, IHC, and IF analyses. RESULTS: The treatment of wogonin attenuated urinary albumin and histopathological damage in tubulointerstitium of diabetic mice. We also found that wogonin down-regulated the expression of pro-inflammatory cytokines and autophagic dysfunction in vivo and in vitro. Molecular docking and Cellular Thermal Shift Assay (CETSA) results revealed that mechanistically phosphoinositide 3-kinase (PI3K) was the target of wogonin. We then found that inhibiting PI3K eliminated the protective effect of wogonin. Wogonin regulated autophagy and inflammation via targeting PI3K, the important connection point of PI3K/Akt/NF-κB signaling pathway. CONCLUSION: Our study is the first to demonstrate the novel role of wogonin in mitigating tubulointerstitial fibrosis and renal tubular cell injury via regulating PI3K/Akt/NF-κB signaling pathway-mediated autophagy and inflammation. Wogonin might be a latent remedial drug against tubular epithelial injury in DN by targeting PI3K.

Predictors of coronary artery calcification and its association with cardiovascular events in patients with chronic kidney disease.

OBJECTIVE: To investigate the predictors of coronary artery calcification (CAC) and its association with cardiovascular events (CVE) in patients with stage 3-5 chronic kidney disease (CKD). METHOD: Two hundred ninety CKD patients in our nephrology department were enrolled from 2018 to May 2019. The levels of matrix Gla protein (MGP) and interleukin 6 (IL-6) were measured via enzyme-linked immunosorbent assay (ELISA) method in 131 CKD patients of all. CAC was evaluated via computed tomography (CT). The covariate factors were analyzed by binary logistic regression analysis. We conducted the visits to explore the prevalence of CVE in 276 CKD patients, and covariate factors were analyzed by Cox proportional hazard model. RESULTS: The prevalence of CAC was up to 57.93%. We found that age, diabetes mellitus, hyperphosphatemia, dialysis duration, and the neutrophil-lymphocyte ratio (NLR) were positively associated with CAC in all patients. In 131 patients, we demonstrated that higher IL-6 and lower MGP levels were associated with CAC. A Cox proportional hazard model demonstrated that moderate to severe CAC was correlated with an increased risk for CVE [Hazard Ratio (HR): 7.250; 95% confidence interval (CI): 3.192-16.470], and a higher MGP level was associated with a reduced risk for CVE (HR: 0.340; 95% CI: 0.124-0.933). CONCLUSIONS: The prevalence of CAC in patients with CKD is a significant issue. Older age, hyperphosphatemia, dialysis duration, diabetes mellitus, IL-6, and the NLR are associated with CAC. In addition, higher MGP levels represent protective factor for CAC. Moderate to severe CAC, and lower MGP levels are associated with an increased risk for CVE. Abbreviations: AGEs: Advanced glycosylation end products; CAC: Coronary artery calcification; CACS: Coronary artery calcification score; Ca: Calcium; CI: confidence interval; CKD: Chronic kidney disease; CVE: Cardiovascular events; CT: Computed tomography; ELISA: Enzyme-linked immunosorbent assay; Hb: hemoglobin; HR: Hazard ratio; hs-CRP: high-sensitivity C-reactive protein; IL-6: Interleukin 6; iPTH: Intact parathyroid hormone; Mg: Magnesium; MGP: Matrix Gla protein; NF-κB: nuclear factor-kappa gene binding; NRL: Neutrophil-lymphocyte ratio; Runx2: Runt-related transcription factor 2; RRT: Renal replacement therapy; P: Phosphorus; Scr: Serum creatinine; TNF--alpha: Tumor necrosis factor--alpha; TC: Total cholesterol; TG: Triglyceride; VSMC: vascular smooth muscle cel.

Preoperative proteinuria may be a risk factor for postoperative acute kidney injury：a meta-analysis.

OBJECTIVE: To investigate the relationship between preoperative proteinuria and postoperative acute kidney injury (AKI). METHODS: We performed a search on databases included PubMed, Embase, the Cochrane Library, and Web of Science, from December 2009 to September 2020. Data extracted from eligible studies were synthesized to calculate the odds ratio (OR) and 95% confidence interval (CI). A fixed or random effects model was applied to calculate the pooled OR based on heterogeneity through the included studies. RESULTS: This meta-analysis of 11 observational studies included 203,987 participants, of whom 21,621 patients suffered from postoperative AKI and 182,366 patients did not suffer from postoperative AKI. The combined results demonstrated that preoperative proteinuria is an independent risk factor for postoperative AKI (adjusted OR = 1.65, 95%CI:1.44-1.89, p < 0.001). Subgroup analysis showed that both preoperative mild proteinuria (adjusted OR = 1.30, 95%CI:1.24-1.36, p < 0.001) and preoperative heavy proteinuria (adjusted OR = 1.93, 95%CI:1.65-2.27, p < 0.001) were independent risk factors for postoperative AKI. The heterogeneity was combined because its values were lower. Further subgroup analysis found that preoperative proteinuria measured using dipstick was an independent risk factor for postoperative AKI (adjusted OR = 1.48, 95%CI:1.37-1.60, p < 0.001). Finally, preoperative proteinuria was an independent risk factor for postoperative AKI in the non-cardiac surgery group (adjusted OR = 2.06, 95%CI:1.31-3.24, p = 0.002) and cardiac surgery group (adjusted OR = 1.69, 95%CI:1.39-2.06, p < 0.001). CONCLUSION: Preoperative proteinuria is an independent risk factor for postoperative AKI and in instances when proteinuria is detected using dipsticks.

Correlation between serum carnosinase concentration and renal damage in diabetic nephropathy patients.

Diabetic nephropathy (DN) is one of the major complications of diabetes and contributes significantly towards end-stage renal disease. Previous studies have identified the gene encoding carnosinase (CN-1) as a predisposing factor for DN. Despite this fact, the relationship of the level of serum CN-1 and the progression of DN remains uninvestigated. Thus, the proposed study focused on clarifying the relationship among serum CN-1, indicators of renal function and tissue injury, and the progression of DN. A total of 14 patients with minimal changes disease (MCD) and 37 patients with DN were enrolled in the study. Additionally, 20 healthy volunteers were recruited as control. Further, DN patients were classified according to urinary albumin excretion rate into two groups: DN with microalbuminuria (n = 11) and DN with macroalbuminuria (n = 26). Clinical indicators including urinary protein components, serum carnosine concentration, serum CN-1 concentration and activity, and renal biopsy tissue injury indexes were included for analyzation. The serum CN-1 concentration and activity were observed to be the highest, but the serum carnosine concentration was the lowest in DN macroalbuminuria group. Moreover, within DN group, the concentration of serum CN-1 was positively correlated with uric acid (UA, r = 0.376, p = 0.026) and serum creatinine (SCr, r = 0.399, p = 0.018) and negatively correlated with serum albumin (Alb, r = - 0.348, p = 0.041) and estimated glomerular filtration rate (eGRF, r = - 0.432, p = 0.010). Furthermore, the concentration of serum CN-1 was discovered to be positively correlated with indicators including 24-h urinary protein-creatinine ratio (24 h-U-PRO/CRE, r = 0.528, p = 0.001), urinary albumin-to-creatinine ratio (Alb/CRE, r = 0.671, p = 0.000), urinary transferrin (TRF, r = 0.658, p = 0.000), retinol-binding protein (RBP, r = 0.523, p = 0.001), N-acetyl-glycosaminidase (NAG, r = 0.381, p = 0.024), immunoglobulin G (IgG, r = 0.522, p = 0.001), cystatin C (Cys-C, r = 0.539, p = 0.001), beta-2-microglobulin (ß2-MG, r = 0.437, p = 0.009), and alpha-1-macroglobulin (α1-MG, r = 0.480, p = 0.004). Besides, in DN with macroalbuminuria group, serum CN-1 also showed a positive correlation with indicators of fibrosis, oxidative stress, and renal tubular injury. Taken together, our data suggested that the level of CN-1 was increased as clinical DN progressed. Thus, the level of serum CN-1 might be an important character during the occurrence and progression of DN. Our study will contribute significantly to future studies focused on dissecting the underlying mechanism of DN.

Carnosine alleviates diabetic nephropathy by targeting GNMT, a key enzyme mediating renal inflammation and fibrosis.

Diabetic nephropathy (DN) is a common microvascular complication of diabetes and the main cause of end-stage nephropathy (ESRD). Inflammation and fibrosis play key roles in the development and progression of diabetic nephropathy. By using in vivo and in vitro DN models, our laboratory has identified the protective role of carnosine (CAR) on renal tubules. Our results showed that carnosine restored the onset and clinical symptoms as well as renal tubular injury in DN. Furthermore, carnosine decreased kidney inflammation and fibrosis in DN mice. These results were consistent with high glucose (HG)-treated mice tubular epithelial cells (MTECs). Using web-prediction algorithms, cellular thermal shift assay (CETSA) and molecular docking, we identified glycine N-methyltransferase (GNMT) as a carnosine target. Importantly, we found that GNMT, a multiple functional protein that regulates the cellular pool of methyl groups by controlling the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), was down-regulated significantly in the serum of Type 1 DM patients and renal tissues of DN mice. Moreover, using cultured TECs, we confirmed that the increased GNMT expression by transient transfection mimicked the protective role of carnosine in reducing inflammation and fibrosis. Conversely, the inhibition of GNMT expression abolished the protective effects of carnosine. In conclusion, carnosine might serve as a promising therapeutic agent for DN and GNMT might be a potential therapeutic target for DN.

Wogonin Ameliorates Renal Inflammation and Fibrosis by Inhibiting NF-κB and TGF-ß1/Smad3 Signaling Pathways in Diabetic Nephropathy.

INTRODUCTION: Diabetic nephropathy (DN) has become an increasing threat to health, and inflammation and fibrosis play important roles in its progression. Wogonin, a flavonoid, has been proven to suppress inflammation and fibrosis in various diseases, including acute kidney injury. This study aimed at investigating the effect of wogonin on diabetes-induced renal inflammation and fibrosis. MATERIALS AND METHODS: Streptozotocin (STZ)-induced diabetic mouse models received gavage doses of wogonin (10, 20, and 40 mg/kg) for 12 weeks. Metabolic indices from blood and urine and pathological damage of glomerulus in the diabetic model were assessed. Glomerular mesangial cells SV40 were cultured in high glucose (HG) medium containing wogonin at concentrations of 1.5825, 3.125, and 6.25 µg/mL for 24 h. Inflammation and fibrosis indices were evaluated by histopathological, Western blotting, and PCR analyses. RESULTS: Wogonin treatment ameliorated albuminuria and histopathological lesions in diabetic mice. Inflammatory cytokines, such as monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and related signaling pathway NF-κB were downregulated after the administration of wogonin in vivo and in vitro. Furthermore, wogonin reduced the expression of extracellular matrix (ECM), including fibronectin (FN), collagen IV (Col-IV), α-smooth muscle actin (α-SMA), and transforming growth factor-ß1 (TGF-ß1) in the kidneys of diabetic mice and HG-induced mesangial cells. Moreover, the inhibition of TGF-ß1/Smad3 pathway might be responsible for these changes. CONCLUSION: Wogonin may ameliorate renal inflammation and fibrosis in diabetic nephropathy by inhibiting the NF-κB and TGF-ß1/Smad3 signaling pathways.

[Effect of electroacupuncture on intestinal Toll-like receptor 4 and nuclear factor-kappa B in obese rats].

OBJECTIVE: To investigate the effect of electroacupuncture (EA) on intestinal Toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) in obese rats, so as to explore the mechanism of action of acupuncture in losing weight. METHODS: A total of 50 male Wistar rats were randomly divided into control and model groups. High-fat feed was used to establish a rat model of obesity, and after modeling, the 24 rats were randomly divided into model group, TLR4 inhibitor group, and EA group, with 8 rats in each group. The rats in the EA group were given EA at "Guanyuan" (CV4), "Zhongwan "(CV12), "Zusanli" (ST36), and" Fenglong" (ST40), 10 minutes each time, 3 times a week, and those in the TLR4 inhibitor group were given intraperitoneal injection of TAK-242 three times a week; the course of treatment was 8 weeks for both groups. Body weight and blood glucose were measured every two weeks. Co-immunoprecipitation was used to observe the interaction between TLR4 and NF-κB p65 in the intestinal tissue; electrophoretic mobility shift assay was used to measure the activity of NF-κB p65; Western blot was used to measure the protein expression of TLR4, phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (p-IκBα), and NF-κB p65; quantitative real-time PCR was used to measure the mRNA expression of TLR4, NF-κB p65, and IκBα. RESULTS: Compared with the control group, the model group had significant increases in body weight, blood glucose, and protein and mRNA expression of TLR4 and NF-κB p65 (P<0.01, P<0.05), as well as significant enhancement in the interaction between TLR4 and NF-κB p65 and activity of NF-κB p65 (P<0.05，P<0.01). Compared with the model group, the EA group had a significant reduction in body weight (P<0.05), both of the EA group and the TLR4 inhibitor group had significant reductions in blood glucose, and protein and mRNA expression of TLR4, p-IκBα， and NF-κB p65 (P<0.05，P<0.01), as well as significant reductions in the activity of NF-κB p65 (P<0.01). CONCLUSION: EA can effectively regulate intestinal TLR4, inhibit the interaction between TLR4 and NF-κB p65, and reduce the activity of NF-κB p65, which may be a potential mechanism of EA in reducing body weight and blood glucose in obese rats.

Rutaecarpine derivative Cpd-6c alleviates acute kidney injury by targeting PDE4B, a key enzyme mediating inflammation in cisplatin nephropathy.

Acute kidney injury (AKI), characterized by a rapid decline in renal function, is triggered by an acute inflammatory response that leads to kidney damage. An effective treatment for AKI is lacking. Using in vitro and in vivo AKI models, our laboratory has identified a series of anti-inflammatory molecules and their derivatives. In the current study, we identified the protective role of rutaecarpine (Ru) on renal tubules. We obtained a series of 3-aromatic sulphonamide-substituted Ru derivatives exhibiting enhanced renoprotective and anti-inflammatory function. We identified Compound-6c(Cpd-6c) as having the best activity and examined its protective effect against cisplatin nephropathy both in vivo and in vitro in cisplatin-stimulated tubular epithelial cells (TECs). Our results showed that Cpd-6c restored renal function more effectively than Ru, as evidenced by reduced blood urea nitrogen and serum creatinine levels in mice. Cpd-6c alleviated tubular injury, as shown by PAS staining and molecular analysis of kidney injury molecule-1 (KIM-1), with both prevention and treatment protocols in cisplatin-treated mice. Moreover, Cpd-6c decreased kidney inflammation, oxidative stress and programmed cell death. These results have also been confirmed in cisplatin-treated TECs. Using web-prediction algorithms, molecular docking, and cellular thermal shift assay (CETSA), we identified phosphodiesterase 4B (PDE4B) as a Cpd-6c target. In addition, we firstly found that PDE4B was up-regulated significantly in the serum of AKI patients. After identifying the function of PDE4B in cisplatin-treated tubular epithelial cells by siRNA transfection or PDE4 inhibitor rolipram, we showed that Cpd-6c treatment did not protect against cisplatin-induced injury in PDE4B knockdown TECs, thus indicating that Cpd-6c exerts its renoprotective and anti-oxidative effects via the PDE4B-dependent pathway. Collectively, Cpd-6c might serve as a potential therapeutic agent for AKI and PDE4B may be highly involved in the initiation and progression of AKI.

RIPK1 inhibitor Cpd-71 attenuates renal dysfunction in cisplatin-treated mice via attenuating necroptosis, inflammation and oxidative stress.

Acute kidney injury (AKI) is a destructive clinical condition induced by multiple insults including ischemic reperfusion, nephrotoxic drugs and sepsis. It is characterized by a sudden decline in renal function, in addition to excessive inflammation, oxidative stress and programmed cell death of renal tubular epithelial cells. RIPK1-mediated necroptosis plays an important role in AKI. In the present study, we evaluated the treatment effects of Compound-71 (Cpd-71), a novel RIPK1 inhibitor, by comparing with Necrostatin-1 (Nec-1), a classic RIPK1 inhibitor, which has several drawbacks like the narrow structure-activity relationship (SAR) profile, moderate potency and non-ideal pharmacokinetic properties, in vivo and in vitro Our results showed that pretreatment of Cpd-71 attenuated cisplatin-induced renal injury, restored renal function and suppressed renal inflammation, oxidative stress and cell necroptosis. In addition, Cpd-71 inhibited renal damage while reducing the up-regulated serum creatinine (Cr) and blood urea nitrogen (BUN) levels in established AKI mice model. Consistently, we confirmed that Cpd-71 exhibited more effectively suppressive effect on cisplatin-induced renal tubular cell necroptosis than Nec-1, by physically binding to the allosteric type III ligand binding site of RIPK1, thereby reduced RIPK1 kinase activity, RIPK1/RIPK3 complex formation and phosphor-MLKL membrane translocation by molecular docking, Western blot, co-immunoprecipitation and cellular thermal shift assay (CETSA). Taken together, we currently showed that targeting RIPK1 with Cpd-71 may serve as a promising clinical candidate for AKI treatment.

Exosomes from high glucose-treated macrophages activate glomerular mesangial cells via TGF-ß1/Smad3 pathway in vivo and in vitro.

Diabetes nephropathy (DN) is characterized by abnormal interactions between kidney-infiltrating macrophages and glomerular mesangial cells. Recently, a novel cell-cell communication mediated by exosomes has gained attention. Exosomes are membrane-bound vesicles that contain a variety of molecules such as proteins, lipids, DNA, mRNA, and microRNAs. Exosomes play an important role in the pathogenesis of DN. In this study, we show that high glucose (HG) led to increased excretion of exosomes from macrophages. Mesangial cells took up exosomes in vitro, which resulted in the activation and proliferation of mesangial cells and the secretion of extracellular matrix and inflammatory cytokines. In addition, C57BL/6 mice injected with exosomes from HG-treated macrophages showed morphologic and functional changes. We then showed that exosomes from HG-treated TGF-ß1 knockdown macrophages induced less extracellular matrix and fewer inflammatory factors in mesangial cells compared with vector control. Our findings suggest that TGF-ß1 mRNA in exosomes serves a role between macrophages and mesangial cells by activating the TGF-ß1/ mothers against decapentaplegic homolog 3 pathway.-Zhu, Q.-J., Zhu, M., Xu, M.-X., Meng, X.-M., Wu, Y.-G. Exosomes from high glucose-treated macrophages activate glomerular mesangial cells via TGF-ß1/Smad3 pathway in vivo and in vitro.