Cullin 3 RING E3 ligase inactivation causes NRF2-dependent NADH reductive stress, hepatic lipodystrophy, and systemic insulin resistance.

Cullin RING E3 ligases (CRL) have emerged as key regulators of disease-modifying pathways and therapeutic targets. Cullin3 (Cul3)-containing CRL (CRL3) has been implicated in regulating hepatic insulin and oxidative stress signaling. However, CRL3 function in liver pathophysiology is poorly defined. Here, we report that hepatocyte Cul3 knockout results in rapid resolution of steatosis in obese mice. However, the remarkable resistance of hepatocyte Cul3 knockout mice to developing steatosis does not lead to overall metabolic improvement but causes systemic metabolic disturbances. Liver transcriptomics analysis identifies that CRL3 inactivation causes persistent activation of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense pathway, which also reprograms the lipid transcriptional network to prevent TG storage. Furthermore, global metabolomics reveals that NRF2 activation induces numerous NAD+-consuming aldehyde dehydrogenases to increase the cellular NADH/NAD+ ratio, a redox imbalance termed NADH reductive stress that inhibits the glycolysis-citrate-lipogenesis axis in Cul3 knockout livers. As a result, this NRF2-induced cellular lipid storage defect promotes hepatic ceramide accumulation, elevates circulating fatty acids, and worsens systemic insulin resistance in a vicious cycle. Hepatic lipid accumulation is restored, and liver injury and hyperglycemia are attenuated when NRF2 activation and NADH reductive stress are abolished in hepatocyte Cul3/Nrf2 double-knockout mice. The resistance to hepatic steatosis, hyperglycemia, and NADH reductive stress are observed in hepatocyte Keap1 knockout mice with NRF2 activation. In summary, our study defines a critical role of CRL3 in hepatic metabolic regulation and demonstrates that the CRL3 downstream NRF2 overactivation causes hepatic metabolic maladaptation to obesity and insulin resistance.

Cullin neddylation inhibitor attenuates hyperglycemia by enhancing hepatic insulin signaling through insulin receptor substrate stabilization.

Hepatic insulin resistance is a hallmark feature of nonalcoholic fatty liver disease and type-2 diabetes and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress-sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin-RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover, but the pathophysiological roles and therapeutic implications of this cellular signaling regulation is largely unknown. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin-like protein called Nedd8 to a cullin scaffold. Here, we report that pharmacological inhibition of cullin neddylation by MLN4924 (Pevonedistat) rapidly decreases hepatic glucose production and attenuates hyperglycemia in mice. Mechanistically, neddylation inhibition delays CRL-mediated IRS protein turnover to prolong insulin action in hepatocytes. In vitro knockdown of either cullin 1 or cullin 3, but not other cullin members, attenuates insulin-induced IRS protein degradation and enhances cellular insulin signaling activation. In contrast, in vivo knockdown of liver cullin 3, but not cullin 1, stabilizes hepatic IRS and decreases blood glucose, which recapitulates the effect of MLN4924 treatment. In summary, these findings suggest that pharmacological inhibition of cullin neddylation represents a therapeutic approach for improving hepatic insulin signaling and lowering blood glucose.

Regioselective and Enantioselective Nickel-Catalyzed Intermolecular Reductive Coupling of Aliphatic Alkenes with Imines.

Unactivated aliphatic alkenes are particularly desirable as starting materials because they are readily accessible in large quantities, but the enantioselective intermolecular reductive coupling of unactivated alkenes with imines is challenging. In this paper, we report a method for nickel-catalyzed intermolecular reductive coupling reactions between aliphatic alkenes and imines to yield chiral amines with excellent enantioselectivities and good linear selectivities. The reaction conditions are compatible with a broad range of aliphatic alkenes, including those derived from bioactive molecules. The success of this method can be attributed to the use of newly developed monodentate chiral spiro phosphine ligands.

Combining ASBT inhibitor and FGF15 treatments enhances therapeutic efficacy against cholangiopathy in female but not male Cyp2c70 KO mice.

Therapeutic reduction of hydrophobic bile acids exposure is considered beneficial in cholestasis. The Cyp2c70 KO mice lack hydrophilic muricholic acids and have a human-like hydrophobic bile acid pool resulting in hepatobiliary injury. This study investigates if combining an apical sodium-dependent bile acid transporter inhibitor GSK2330672 (GSK) and fibroblast growth factor-15 (FGF15) overexpression, via simultaneous inhibition of bile acid synthesis and gut bile acid uptake, achieves enhanced therapeutic efficacy in alleviating hepatobiliary injury in Cyp2c70 KO mice. The effects of GSK, adeno-associated virus (AAV)-FGF15, and the combined treatment on bile acid metabolism and cholangiopathy were compared in Cyp2c70 KO mice. In female Cyp2c70 KO mice with more severe cholangiopathy than male Cyp2c70 KO mice, the combined treatment was more effective in reversing portal inflammation, ductular reaction, and fibrosis than AAV-FGF15, while GSK was largely ineffective. The combined treatment reduced bile acid pool by ∼80% compared to ∼50% reduction by GSK or AAV-FGF15, and enriched tauro-conjugated ursodeoxycholic acid in the bile. Interestingly, the male Cyp2c70 KO mice treated with AAV-FGF15 or GSK showed attenuated cholangiopathy and portal fibrosis but the combined treatment was ineffective despite reducing bile acid pool. Both male and female Cyp2c70 KO mice showed impaired gut barrier integrity. AAV-FGF15 and the combined treatment, but not GSK, reduced gut exposure to lithocholic acid and improved gut barrier function. In conclusion, the combined treatment improved therapeutic efficacy against cholangiopathy than either single treatment in the female but not male Cyp2c70 KO mice by reducing bile acid pool size and hydrophobicity.

Organophosphine as an Alkyl Transfer Shuttle for the Direct ß-Alkylation of Chalcones Using Alkyl Halides.

We report an efficient alkyl transfer strategy for the direct ß-alkylation of chalcones using commercially available alkyl bromides as alkyl reagents. In this transformation, the ortho-phosphanyl substituent in the chalcones is crucial for controlling their reactivity and selectivity. It also serves as a reliable alkyl transfer shuttle to transform electrophilic alkyl bromides into nucleophilic alkyl species in the form of quaternary phosphonium salts and transfer the alkyl group effectively to the ß-position of the chalcones. This alkyl transfer strategy can be further extended to the alkenylation of ortho-phosphanyl benzaldehydes to assemble functionalized polyenes.

EZH2 promotes angiogenesis in peritoneal dialysis by epigenetically activating SP4 expression in the IL-6/sIL-6R signalling pathway.

Background: Interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R) promotes peritoneal angiogenesis by stimulating SP4-mediated vascular endothelial growth factor (VEGF) production in peritoneal dialysis (PD). Moreover, histone methyltransferase enhancer of zeste homologue 2 (EZH2) is involved in IL-6/sIL-6R signalling via the acceleration of vascular endothelial growth factor (VEGF)-induced angiogenesis. However, the molecular mechanism underlying how EZH2 epigenetically activates VFGF expression in IL-6/sIL-6R signalling during PD is still unclear. Methods and Results: In this study, we measured the expression of EZH2, DNMT3B and SP4 in human peritoneal mesothelial cells (HPMCs) treated with IL-6/sIL-6R stimulation and/or EZH2 overexpression, silencing or inhibition. Methylation of the CpG site in the SP4 promoter region and VEGF production were measured under these treatments in HPMCs. Moreover, tube formation in human umbilical vein endothelial cells (HUVECs) was detected following treatment with conditioned media from these stimulated HPMCs. The 5/6 nephrectomy (5/6Nx) rat model was established, and the rats were injected with peritoneal dialysate. EZH2, DNMT3B and SP4 expression and microvessels were analysed in 5/6Nx + PD rats treated with IL-6/sIL-6R and EZH2 overexpression. The results showed that IL-6/sIL-6R and EZH2 overexpression enhanced the expression of EZH2, DNMT3B and SP4, but EZH2 silencing/inhibition reduced these expression levels. The results for VEGF production and tube formation in vitro followed the same trend. IL-6/sIL-6R and EZH2 overexpression increased the methylation percentage of the -170 bp CpG site in the SP4 promoter region in HPMCs. Moreover, IL-6/sIL-6R and EZH2 overexpression stimulated EZH2, DNMT3B and SP4 expression and promoted angiogenesis in 5/6Nx + PD rats. Conclusions: Thus, this study indicated that EZH2 is involved in IL-6/sIL-6R signalling and epigenetically regulates SP4 expression, thereby stimulating VEGF production and angiogenesis in PD. Targeting EZH2 is expected to be a novel therapeutic approach for end-stage renal disease (ESRD) patients with PD treatment.

Baseline anxiety disorders are associated with progression of diabetic kidney disease in type 2 diabetes.

BACKGROUND: Diabetic kidney disease (DKD) is a leading cause of kidney failure worldwide. Anxiety has been associated with disease progression in non-diabetes patients. We aimed to examine the prospective association between anxiety and progression of DKD in type 2 diabetes. METHODS: We conducted a prospective cohort study of 2040 participants with type 2 diabetes at the Diabetes Center of Shanghai General Hospital between May 2017 and June 2020. Anxiety disorders at baseline were diagnosed by a structured clinical interview based on the 10th Revision of International Classification of Disease (ICD). Progression of DKD was identified as the transition from one urinary albumin excretion rate (AER) stage to the next or the development of kidney failure during the follow-up period. RESULTS: At baseline, 403 (19.8%) had a diagnosis of anxiety disorders, of whom 107 (26.6%) also received a depression diagnosis. During a median follow-up time of 3.2 years, deterioration of the kidney status occurred in 340 (16.7%) individuals. After adjustment for potential confounders including depression or an anxiety × depression interaction term, anxiety disorders were independently related to an increased risk of progression of DKD (HR 1.539, 95% CI 1.130-2.095, p = 0.006; HR 1.536, 95% CI 1.111-2.122, p = 0.009, respectively). CONCLUSIONS: Anxiety disorders at baseline, independent of possible confounders, were associated with the progression of DKD in type 2 diabetes. Whether therapeutic interventions for anxiety reduce the risk needs to be investigated.

Association of serum thymosin ß4 with malnutrition-inflammation-atherosclerosis syndrome in peritoneal dialysis patients: a cross-sectional study.

BACKGROUND: Malnutrition-inflammation-atherosclerosis (MIA) syndrome may worsen the prognosis of peritoneal dialysis (PD) patients. Serum thymosin ß4 (sTß4) protects against inflammation, fibrosis and cardiac dysfunction. OBJECTIVES: The present study aimed to characterize the association between sTß4 and MIA syndrome as well as to investigate the potential of regulating sTß4 to improve the prognosis of PD patients. METHODS: We performed a cross-sectional, single-center pilot study involving 76 PD patients. Demographic characteristics, clinical characteristics, nutritional profiles, inflammatory mediators, atherosclerosis-related factors and sTß4 levels were collected and subjected to association analysis for sTß4 and MIA syndrome. RESULTS: sTß4 levels did not significantly vary with sex or primary disease in PD patients. Ages and PD features did not vary between patients with different levels of sTß4. PD patients with higher levels of sTß4 had significantly higher levels of nutritional indicators, including subjective global nutritional assessment (SGA) (p < 0.001) and serum albumin (ALB) (p < 0.001) but lower levels of inflammatory and atherosclerotic indicators, including serum C reaction protein (CRP) (p = 0.009), the right common carotid artery (RCCA) intimal thickness (p < 0.001) and the left common carotid artery (LCCA) intimal thickness (p = 0.02). Correlation analysis showed that sTß4 was positively associated with SGA (p < 0.001) and serum ALB (p < 0.001) but negatively associated with CRP (p = 0.020), RCCA intimal thickness (p < 0.001) and LCCA intimal thickness (p = 0.033). In multiple adjusted models, the prevalence of MIA syndrome was significantly decreased in PD patients with increased levels of sTß4 when patients without MIA syndrome were compared to those with all indicators of MIA syndrome (OR = 0.996, 95% CI 0.993-0.999, p = 0.003) or those with at least one indicator of MIA syndrome (OR = 0.997, 95% CI 0.995-0.998, p < 0.001). CONCLUSIONS: The sTß4 level decreases in PD patients with MIA syndrome. The prevalence of MIA syndrome decreases significantly as the level of sTß4 increases in PD patients.

Choosing an appropriate glomerular filtration rate estimating equation: role of body mass index.

BACKGROUND: We aimed to investigate the accuracy of different equations in evaluating estimated glomerular filtration rate (eGFR) in a Chinese population with different BMI levels. METHODS: A total of 837 Chinese patients were enrolled, and the eGFRs were calculated by three Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations, three full-age spectrum (FAS) equations and two Modification of Diet in Renal Disease (MDRD) equations. Results of measured GFR (mGFR) by the 99Tcm-diathylenetriamine pentaacetic acid (99Tcm-DTPA) renal dynamic imaging method were the reference standards. According to BMI distribution, the patients were divided into three intervals: below 25th(BMIP25), 25th to 75th(BMIP25-75) and over 75th percentiles (BMIP75). RESULTS: The medium BMI of the three BMI intervals were 20.9, 24.8 and 28.9 kg/m2, respectively. All deviations from mGFR (eGFR) were correlated with BMI (p < 0.05). The percentage of cases in which eGFR was within mGFR ±30% (P30) was used to represent the accuracy of each equation. Overall, eGFRFAS_Cr_CysC and eGFREPI_Cr_2009 performed similarly, showing the best agreement with mGFR among the eight equations in Bland-Altman analysis (biases: 4.1 and - 4.2 mL/min/1.73m2, respectively). In BMIP25 interval, eGFRFAS_Cr got - 0.7 of the biases with 74.2% of P30, the kappa value was 0.422 in classification of CKD stages and the AUC60 was 0.928 in predicting renal insufficiency, and eGFREPI_Cr_2009 got 2.3 of the biases with 71.8% of P30, the kappa value was 0.418 in classification of CKD stages and the AUC60 was 0.920 in predicting renal insufficiency. In BMIP25-75 interval, the bias of eGFRFAS_Cr_CysC was 4.0 with 85.0% of P30, the kappa value was 0.501 and the AUC60 was 0.941, and eGFRFAS_Cr_CysC showed balanced recognition ability of each stage of CKD (62.3, 63.7, 68.0, 71.4 and 83.3% respectively). In BMIP75 interval, the bias of eGFREPI_Cr_CysC_2012 was 3.8 with 78.9% of P30, the kappa value was 0.484 the AUC60 was 0.919, and eGFREPI_Cr_CysC_2012 equation showed balanced and accurate recognition ability of each stage (60.5, 60.0, 71.4, 57.1 and 100% respectively). In BMIP75 interval, the bias of eGFRFAS_Cr_CysC was - 1.8 with 78.5% of P30, the kappa value was 0.485, the AUC60 was 0.922. However, the recognition ability of each stage of eGFRFAS_Cr_CysC eq. (71.1, 61.2, 70.0, 42.9 and 50.0% respectively) was not as good as GFREPI_Cr_CysC_2012 equation. CONCLUSION: For a Chinese population, we tend to recommend choosing eGFRFAS_Cr and eGFREPI_Cr_2009 when BMI was around 20.9, eGFRFAS_Cr_CysC when BMI was near 24.8, and eGFREPI_Cr_CysC_2012 when BMI was about 28.9.

Short-term prognosis and influencing factors of patients with acute kidney injury treated with prolonged intermittent renal replacement therapy.

BACKGROUND: Studies assessing prognosis after prolonged intermittent renal replacement therapy (PIRRT) for acute kidney injury (AKI) are scarce. AIM: To assess the impact of PIRRT on AKI and factors associated with short-term prognosis. METHODS: In this retrospective nested case-control study, AKI patients administered PIRRT in Shanghai General Hospital from 01/2012 to 10/2018 were assigned to the 30-day survivor and death groups. Surviving patients were further divided into the kidney recovery and non-recovery groups at 30 and 90 days post-discharge, respectively. Propensity score matching was performed. RESULTS: Totally 576 patients were included in the non-matched study population, mortality and kidney recovery rates were 51.7% and 33.4%, respectively. After propensity score matching, there were 250 patients in each of the death and survival groups. Low PIRRT frequency (OR = 2.165, 95% CI = 1.178-3.978), infection (OR = 0.447, 95% CI = 0.251-0.795), number of damaged vital organs (OR = 0.478, 95% CI = 0.346-0.661), sodium (OR = 0.958, 95% CI = 0.928-0.988), total protein (OR = 1.047, 95% CI = 1.022-1.072), pre-dialysis thrombin time (TT; OR = 0.959, 95% CI = 0.936-0.983), pre-discharge glomerular filtration rate (GFR; OR = 1.024, 95% CI = 1.017-1.031) and admission ward [reference: renal ward; intensive care unit (OR = 0.042, 95% CI = 0.008-0.211); surgery (OR = 0.092, 95% CI = 0.018-0.465); medical (OR = 0.049, 95% C% CI = 0.009-0.259); other (OR = 0.097, 95% CI = 0.016-0.572)] independently predicted 30-day mortality. Peripherally inserted central catheter (OR = 13.970, 95% CI = 1.439-135.589), urea nitrogen (OR = 0.961, 95% CI = 0.933-0.990) and pre-discharge GFR (OR = 1.102, 95% CI = 1.067-1.137) independently predicted 30-day kidney recovery. Pre-dialysis Scr (OR = 0.997, 95% CI = 0.995-0.999), urea nitrogen (OR = 0.948, 95% CI = 0.912-0.986) and pre-discharge GFR (OR = 1.137 95% CI = 1.088-1.189) independently predicted 90-day kidney recovery. CONCLUSIONS: PIRRT improves survival and kidney function recovery in AKI patients. In patients with previous GFR ≥ 30 mL/(min-1.73 m2 ) and no prior maintenance dialysis, PIRRT at 3-5 sessions/week might be appropriate.

Endothelin-1 triggers human peritoneal mesothelial cells' proliferation via ERK1/2-Ets-1 signaling pathway and contributes to endothelial cell angiogenesis.

BACKGROUND: Dysfunction of the peritoneum as a dialysis organ may result from progressive membrane injury on peritoneal dialysis (PD). It has been increasingly recognized that the human peritoneal mesothelial cells (HPMCs) play a key role in peritoneal membrane early injury. Recently, it has been reported that bioincompatible PD fluid with high concentrations of glucose and glucose degradation products, low pH, high osmolality, and peritonitis stimulates HPMCs to release endothelin-1 (ET-1). ET-1 causes increased the release of vascular endothelial growth factor, which is important for tumor cell angiogenesis. We hypothesized that activating ET-1 might predict injury of peritoneal membrane. METHODS: HPMCs were isolated from normal omentum. ERK1/2 and Ets-1 phosphorylation were measured by Western blot analysis. HPMC proliferation was detected by the bromodeoxyuridine (BrdU) assay. Capillary networks of tubes formed were photographed under a microscope, and five randomly selected fields from each well were analyzed for total capillary length by using Image J software. RESULTS: MEK-1 blocker significantly abolished the ERK1/2 activation by ET-1, which also triggered phosphorylation, thus activating the transcription factor Ets-1 downstream from ERK1/2. ET-1 was capable to induce HPMC proliferation, which could be attenuated by ET-1 antagonists. Antibody and small interfering RNA mediated blockade of Ets-1 had similar antiproliferative effects. Thus, ET-1 specifically triggered HPMC proliferation via ERK1/2-Ets-1 signaling pathway. VEGF production and endothelial cell angiogenesis were significantly in response to conditioned medium from HPMCs treated with ET-1. CONCLUSIONS: ET-1 triggered HPMC proliferation through the ERK1/2-Ets-1 signaling pathway and contributed to VEGF production and endothelial cell angiogenesis.

Phosphate stimulates myotube atrophy through autophagy activation: evidence of hyperphosphatemia contributing to skeletal muscle wasting in chronic kidney disease.

BACKGROUND: Accelerated muscle atrophy is associated with a three-fold increase in mortality in chronic kidney disease (CKD) patients. It is suggested that hyperphosphatemia might contribute to muscle wasting, but the underlying mechanisms remain unclear. Although evidence indicates that autophagy is involved in the maintenance of muscle homeostasis, it is not known if high phosphate levels can result in activation of autophagy, leading to muscle protein loss. METHODS: Immortalized rat L6 myotubes were exposed to a high concentration of phosphate, with or without autophagy inhibition. Myotube atrophy was examined by phase contrast microscopy. Autophagic activity was assessed by measuring the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 using quantitative real-time polymerase chain reaction and western blot. RESULTS: Phosphate induced cell atrophy in L6 myotubes in a dose- and time-dependent manner, and these responses were not associated with calcification or osteogenesis. Phosphate also dose- and time-dependently increased the LC3-II/LC3-I ratio. Inhibition of autophagy with wortmannin or knockdown of Atg5 significantly suppressed myotube atrophy caused by high phosphate concentration. CONCLUSIONS: High phosphate concentration induces muscle cell atrophy through the activation of autophagy. Targeting autophagy could be a therapeutic strategy for preventing muscle wasting caused by hyperphosphatemia.

Hepatitis B virus X protein modulates renal tubular epithelial cell-induced T-cell and macrophage responses.

Renal tubular epithelial cells (RTECs) have an active role in renal inflammation, functioning as antigen-presenting cells as they constitutively express major histocompatibility complex-II and co-stimulatory molecules that can activate T cells and macrophages. Previous studies indicate that inflammatory cell infiltration and tubulointerstitial fibrosis are present in renal biopsies from Hepatitis B virus (HBV)-associated glomerulonephritis (HBV-GN) patients. We hypothesized that disorder RTECs may be involved in the progression of HBV-GN. Here, we measured renal function and inflammatory cells infiltration in C57BL/6J-TgN mice, and data showed that in C57BL/6J-TgN mice HBV x protein (HBx) mainly deposited in RTECs, and CD4(+) T cells and macrophages infiltrated into the interstitium. In vitro HBx upregulated CD40 expression in RTECs. In HK-2/CD4(+) T cells co-culture system, we found that HBx-stimulated HK-2 cells could activate CD4(+) T cells, promote their proliferation, and lead to an imbalance of interleukin (IL)-4 and interferon-γ. In HK-2/macrophages co-culture system, we found that HBx-stimulated HK-2 cells also increased macrophage adherent capacity and promoted MCP-1 and tumor necrosis factor-α and IL-1ß secretion. These immune dysfunction may contribute to the pathogenesis of HBV-GN.

Vitamin D supplements in chronic kidney disease.

Chronic kidney disease (CKD) is a significant public health problem and Vitamin D deficiency is prevalent in CKD and might be associated with calcium and phosphate metabolism, cardiovascular disease, infections as well as the progress of kidney dysfunction. Emerging evidence implies that Vitamin D supplements may be of benefit to CKD. Based on existing laboratory and clinical evidence, this review intends to discuss the effectiveness of Vitamin D supplements and controversy in clinical practice. The effect of Vitamin D in CKD patients is summarized in detail from CKD-mineral bone disease, the progression of renal function, cardiovascular events and immune system. Considerable disputes exist for the Vitamin D supplements in CKD, and a growing amount of experimental evidence and some clinical evidence are now gathering from in vitro, animal and epidemiological studies.

[The role of Foxo1 and ubiquitin-proteasome system markers in muscle atrophy caused by chronic kidney disease].

OBJECTIVE: To identity whether there is muscle atrophy phenomenon in end-stage kidney disease patients and to detect the level of transcription factor Foxo1 and the activity of ubiquitin-proteasome system. METHODS: Twenty-two patients in chronic kidney disease (CKD) stage 5 were selected and their mean muscle cross sectional area was measured. mRNA and protein levels of Foxo1, Atrogin-1, MuRF1 in rectus abdominis biopsies obtained from consecutive patients were detected. Control biopsies were obtained from 8 healthy subjects during elective surgery for abdominal wall hernias and 6 subjects during elective surgery for adenomyosis. RESULTS: Compared with the control group, cross sectional area of muscle fibers decreased and the transcription and protein levels of Foxo1, Atrogin-1, MuRF1 were upregulated in CKD group (P < 0.05). Protein level of p-Foxo1 decreased in CKD group (P < 0.05). CONCLUSION: There exist muscle atrophy phenomenon in CKD patients, which may associate with the upregulation of Foxo1 and activation of ubiquitin-proteasome system.

Bile acids regulation of cellular stress responses in liver physiology and diseases.

Bile acids are physiological detergents and signalling molecules that are critically implicated in liver health and diseases. Dysregulation of bile acid homeostasis alters cell function and causes cell injury in chronic liver diseases. Therapeutic agents targeting bile acid synthesis, transport and signalling hold great potential for treatment of chronic liver diseases. The broad cellular and physiological impacts of pharmacological manipulations of bile acid metabolism are still incompletely understood. Recent research has discovered new links of bile acid signalling to the regulation of autophagy and lysosome biology, redox homeostasis and endoplasmic reticulum stress. These are well-conserved mechanisms that allow cells to adapt to nutrient and organelle stresses and play critical roles in maintaining cellular integrity and promoting survival. However, dysregulation of these cellular pathways is often observed in chronic liver diseases, which exacerbates cellular dysfunction to contribute to disease pathogenesis. Therefore, identification of these novel links has significantly advanced our knowledge of bile acid biology and physiology, which is needed to understand the contributions of bile acid dysregulation in disease pathogenesis, establish bile acids as diagnostic markers and develop bile acid-based pharmacological interventions. In this review, we will first discuss the roles of bile acid dysregulation in the pathogenesis of chronic liver diseases, and then discuss the recent findings on the crosstalk of bile acid signalling and cellular stress responses. Future investigations are needed to better define the roles of these crosstalks in regulating cellular function and disease processes.

Deletion of hepatocyte cysteine dioxygenase type 1, a bile acid repressed gene, enhances glutathione synthesis and ameliorates acetaminophen hepatotoxicity.

Liver is a major organ that metabolizes sulfur amino acids cysteine, which is the substrate for the synthesis of many essential cellular molecules including GSH, taurine, and coenzyme A. Bile acid-activated farnesoid x receptor (FXR) inhibits cysteine dioxygenase type 1 (CDO1), which mediates hepatic cysteine catabolism and taurine synthesis. To define the impact of bile acid inhibition of CDO1 on hepatic sulfur amino acid metabolism and antioxidant capacity, we developed hepatocyte-specific CDO1 knockout mice (Hep-CDO1 KO) and hepatocyte specific CDO1 transgenic mice (Hep-CDO1 Tg). Liver metabolomics revealed that genetic deletion of hepatic CDO1 reduced de novo taurine synthesis but had no impact on hepatic taurine abundance or bile acid conjugation. Consistent with reduced cysteine catabolism, Hep-CDO1 KO mice showed increased hepatic cysteine abundance but unaltered methionine cycle intermediates and coenzyme A synthesis. Upon acetaminophen overdose, Hep-CDO1 KO mice showed increased GSH synthesis capacity and alleviated liver injury. In contrast, hepatic CDO1 overexpression in Hep-CDO1 Tg mice stimulated hepatic cysteine to taurine conversion, resulting in reduced hepatic cysteine abundance. However, Hep-CDO1 Tg mice and WT showed similar susceptibility to acetaminophen-induced liver injury. Hep-CDO1 Tg mice showed similar hepatic taurine and coenzyme A compared to WT mice. In summary, these findings suggest that bile acid and FXR signaling inhibition of CDO1-mediated hepatic cysteine catabolism preferentially modulates hepatic GSH synthesis capacity and antioxidant defense, but has minimal effect on hepatic taurine and coenzyme A abundance. Repression of hepatic CDO1 may contribute to the hepatoprotective effects of FXR activation under certain pathologic conditions.

The role of the toll-like receptor TLR4 in hepatitis B virus-associated glomerulonephritis.

Toll-like receptor 4 (TLR4) plays an important role in innate immunity. The aim of our study was to detect the expression of TLR4 in HBV-associated glomerulonephritis (HBV-GN) and explore the pathogenesis of TLR4 in inhibition of HBV replication in kidney. Immunohistochemical methods were used to detect the distribution of immunoglobulin, HBsAg, HBcAg and TLR4. Because TLR4 was mainly distributed in renal tubules and interstitial spaces, we used human proximal tubular epithelial cells (HK-2) as target cells, which were cultured with HBV-DNA-positive serum. Cells were divided into four groups: A, a normal control group; B, an HBV-induced group; C, an HBV and 10 µg/ml LPS (TLR4-stimulating factor) group; and D, an HBV and 5 µg/ml CLI095 (TLR4 inhibitor) group. The morphology of HK-2 cells was observed by microscopy, and the expression of α-SMA was examined by immunofluorescence before and after culturing with HBV-DNA-positive serum. MTT was used to detect HK2 proliferation. The expression of TLR4 protein was detected by immunofluorescence and western-blotting assays, HBsAg and HBeAg levels in supernatants were measured by ELISA, and the expression of HBV-DNA was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The immunohistochemical staining for TLR4 in the normal control group was negative. The expression of TLR4 in HBV-GN was significantly higher than in the HBV-antigen-positive and -negative primary glomerulonephritis (PGN) groups and was mainly distributed in renal tubules and the interstitial area, where the distribution of HBsAg had similar intensity. The expression of TLR4 was significantly associated with renal tubular and interstitial lesions. In an in vitro study with prolonged infection with HBV-DNA-positive serum, more irregularly shaped cells and fewer HK-2 were observed. The expression of α-SMA in the cytoplasm of HK-2 was significantly increased after HBV infection. Immunofluorescence results showed that almost no expression of TLR4 in HK2 cells cultured with normal serum (group A), while the expression of TLR4 was increased after HBV infection (group B). With increasing LPS concentration, the rate of the proliferation of HK2 cells, the levels of HBV-DNA in the supernatant, and the expression of HBsAg and HBeAg decreased. TLR4 was mainly expressed in HBV-GN in renal tubules and interstitial spaces and was significantly associated with renal tubular and interstitial lesions. The stimulation of TLR4 not only inhibited HBV replication but also inevitably induced immune injury to cells. Therefore, we speculate that TLR4 may be involved in an immune inflammatory reaction by inhibiting HBV replication in HK2 cells, which could have an antiviral effect during HBV infection in the kidney.

[Related factors and prognostic significance of intradialytic blood pressure variability in patients on maintenance hemodialysis].

OBJECTIVE: To evaluate intradialytic blood pressure variability (BPV) in patients on maintenance hemodialysis (MHD), and to investigate the correlated factors of BPV in MHD process and its correlation with prognosis. METHODS: Patients with end stage renal disease on MHD before January 1, 2009 were enrolled and analyzed retrospectively. Blood pressure at the first hemodialysis every quarter during January, 2009 and December, 2010 were recorded. The systolic pressure, diastolic pressure were calculated, and dialysis systolic and diastolic BPV were expressed with discrete coefficients. As for patients with follow-up time less than 2 years, blood pressures in evenly distributed 6-8 courses were used for calculation.Cardiovascular events and death were recorded and the follow-up was lasted till December 31, 2011. RESULTS: A total of 280 patients were enrolled, with intradialytic systolic BPV of 0.119 ± 0.029, and intradialytic diastolic BPV of 0.118 ± 0.028. Intradialytic systolic BPV in the elderly group (n = 114) was significantly higher than that in the younger group (n = 166) (0.126 ± 0.029 vs 0.114 ± 0.028, P = 0.012), while no significant difference was found in diastolic BPV (0.117 ± 0.031 vs 0.119 ± 0.025, P = 0.498). Intradialytic systolic BPV was used as variates in multivariable regression analysis, and results showed that age, systolic blood pressure before dialysis, intradialytic weight gain (IDWG) rate during dialysis and hemoglobin level were independent influential factors for intradialytic systolic BPV. The intradialytic diastolic BPV was used as variates in multivariable regression analysis, and results showed that IDWG rate and average dehydration volume were independent influential factors for intradialytic diastolic BPV. During 3 years of follow-up, 64 patients died (22.9%). The survival analysis showed that the dialysis systolic BPV elevation was associated with the mortality rate (P < 0.01). CONCLUSIONS: Older age, high systolic pressure before hemodialysis, high IDWG rate, and low hemoglobin level were independent risk factors of high intradialytic systolic BPV increase. Intradialytic high IDWG is an independent risk factor of high intradialytic diastolic BPV increase in patients on MHD. Intradialytic systolic BPV increase is associated with all-cause mortality in patients on MHD.

[Autophagy-lysosome pathway in skeletal muscle of diabetic nephropathy rats and the effect of low-protein diet plus α-keto acids on it].

OBJECTIVE: To explore the regulation of autophagy-lysosome pathway (ALP) in skeletal muscle of diabetic nephropathy and examine the effect of low protein diet plus α-keto acid on ALP. METHODS: A total of 45 24-week-old Goto-Kakizaki rats were randomized to receive normal protein (22%) diet (NPD), low-protein (6%) diet (LPD) or low-protein (5%) plus α-keto acids (1%) diet (Keto) (n = 15 each). Wistar control rats had a normal protein diet. The mRNA and protein levels of ALP markers LC3B, Bnip3, Cathepsin L in soleus muscle were evaluated at 48 weeks. Electron microscopy was used to confirm the changes of autophagy. RESULTS: Compared with CTL group, the mRNA levels of LC3B, Bnip3, Cathepsin L in soleus muscle of rats on NPD were higher, and protein levels of LC3B-I, LC3B-II, Bnip3, Cathepsin L in soleus muscle of rats on NPD also higher than CTL group (0.82 ± 0.33 vs 0.25 ± 0.07, 0.76 ± 0.38 vs 0.20 ± 0.12, 1.25 ± 0.30 vs 0.56 ± 0.19, 1.29 ± 0.40 vs 0.69 ± 0.20). The mRNA levels of LC3B, Bnip3 and Cathepsin L in LPD group were slightly lower, compared with NPD group. However there was no statistical significance. Similarly the protein levels of LC3B-I, LC3B-II, Bnip3 and Cathepsin L in LPD group were slightly lower with no statistical significance. In contrast, the mRNA levels of LC3B, Bnip3 and Cathepsin L were greatly lower in Keto group in comparison with NPD and LPD. And protein levels of LC3B-I, LC3B-II, Bnip3 and Cathepsin L were also greatly lower in Keto group in comparison with NPD and LPD. Additionally, autophagosome or auto-lysosome was found in NPD and LPD groups by electron microscopy. CONCLUSIONS: ALP is activated in skeletal muscle of diabetic nephropathy rats. And low protein plus α-keto acid decrease the activation of ALP and improve muscle wasting.