The role of KLF2 in regulating hepatic lipogenesis and blood cholesterol homeostasis via the SCAP/SREBP pathway.

Liver steatosis is a common metabolic disorder resulting from imbalanced lipid metabolism, which involves various processes such as de novo lipogenesis, fatty acid uptake, fatty acid oxidation, and VLDL secretion. In this study, we discovered that KLF2, a transcription factor, plays a crucial role in regulating lipid metabolism in the liver. Overexpression of KLF2 in the liver of db/db mice, C57BL/6J mice, and Cd36-/- mice fed on a normal diet resulted in increased lipid content in the liver. Additionally, transgenic mice (ALB-Klf2) that overexpressed Klf2 in the liver developed liver steatosis after being fed a normal diet. We found that KLF2 promotes lipogenesis by increasing the expression of SCAP, a chaperone that facilitates the activation of SREBP, the master transcription factor for lipogenic gene expression. Our mechanism studies revealed that KLF2 enhances lipogenesis in the liver by binding to the promoter of SCAP and increasing the expression of genes involved in fatty acid synthesis. Reduction of KLF2 expression led to a decrease in SCAP expression and a reduction in the expression of SREBP1 target genes involved in lipogenesis. Overexpression of KLF2 also increased the activation of SREBP2 and the mRNA levels of its downstream target SOAT1. In C57BL/6J mice fed a high-fat diet, overexpression of Klf2 increased blood VLDL secretion, while reducing its expression decreased blood cholesterol levels. Our study emphasizes the novelty that hepatic KLF2 plays a critical role in regulating lipid metabolism through the KLF2/SCAP/SREBPs pathway, which is essential for hepatic lipogenesis and maintaining blood cholesterol homeostasis.

CD36 regulates macrophage and endothelial cell activation and multinucleate giant cell formation in anti neutrophil cytoplasm antibody vasculitis.

OBJECTIVE: To investigate CD36 in ANCA-associated vasculitis (AAV), a condition characterized by monocyte/macrophage activation and vascular damage. METHODS: CD36 expression was assessed in AAV patients and healthy controls (HC). The impact of palmitic acid (PA) stimulation on multinucleate giant cell (MNGC) formation, macrophage, and endothelial cell activation, with or without CD36 knockdown, was examined. RESULTS: CD36 was overexpressed on AAV patients' monocytes compared to HC, regardless of disease activity. AAV patients exhibited elevated soluble CD36 levels in serum and plasma and PR3-ANCA patients' monocytes demonstrated increased MNGC formation following PA stimulation compared to HC. PA stimulation of macrophages or endothelial cells resulted in heightened CD36 expression, cell activation, increased macrophage migration inhibitory factor (MIF) production, and c-Myc expression, with attenuation upon CD36 knockdown. CONCLUSION: CD36 participates in macrophage and endothelial cell activation and MNGC formation, features of AAV pathogenesis. AAV treatment may involve targeting CD36 or MIF.

The ratio of neutrophils to lymphocytes effectively predicts clinical outcomes in idiopathic membranous nephropathy.

BACKGROUND: Systemic inflammatory indicators are important in the prognoses of various diseases. Such indicators, including the neutrophil-to-lymphocyte ratio (NLR), can be meaningful in predicting the clinical outcome in patients diagnosed with idiopathic membranous nephropathy (IMN). MATERIALS AND METHODS: 112 IMN patients diagnosed by renal biopsy were recruited retrospectively. The endpoint was defined as a combination of partial and complete remission. Statistical analysis determined the independent factors associated with clinical remission and the predictive utility of NLR. RESULTS: Within the 12-month follow-up period, 72 patients achieved clinical remission after treatment. Univariate analysis identified significant differences in serum albumin, estimated glomerular filtration rate (eGFR), proteinuria, neutrophil count, and NLR between the remission group and the non-remission group (all p < 0.05). Cox proportional hazards indicated that elevated eGFR (HR 1.022, 95% CI (1.009 - 1.035), p = 0.001), lower NLR (HR 0.345, 95% CI (0.237 - 0.501), p = 0.0001), and decreased proteinuria (HR 0.826, 95% CI (0.693 - 0.984), p = 0.032) were protective elements for remission. With an optimal cut-off value of 2.61, the pre-treatment NLR had an excellent ability to identify the remission (area under the curve (AUC), 0.785). Participants were separated into low- and high-NLR groups by using 2.61. Kaplan-Meier survival curves revealed significantly higher remission rates in the lower group (p < 0.0001). CONCLUSION: The NLR is an effective indicator for predicting clinical remission in patients with IMN.

Roxadustat Versus Erythropoietin: The Comparison of Efficacy in Reversing Ventricular Remodeling in Dialysis Patients with Anaemia.

Background: Renal anaemia and left ventricular hypertrophy are the main complications of chronic kidney disease and are shared among dialysis patients. This retrospective study aimed to compare the efficacies of the hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat and recombinant human erythropoietin in reversing ventricular remodeling in dialysis patients with renal anaemia. Methods: A total of 204 participants underwent baseline examinations, including echocardiograms and laboratory tests, before being administered either treatment for at least 24 weeks from January 2018 to October 2021, after which follow-up examinations were conducted at 6 months. Propensity score matching based on key variables included age, gender, cardiovascular diseases, cardiovascular medications, dialysis course and the vascular access at baseline was performed to include populations with similar characteristics between groups. Results: In total, 136 patients were included with roxadustat or recombinant human erythropoietin. The left ventricular mass index after treatment with roxadustat and recombinant human erythropoietin both significantly decreased after 6 months, but there was no significant difference in the change in left ventricular mass index between the two groups. In addition, the left ventricular end-diastolic diameters and left ventricular wall thickness, systolic blood pressure, and diastolic blood pressure significantly decreased in the roxadustat group. Roxadustat and recombinant human erythropoietin also increased haemoglobin significantly, but there was no significant difference in the change in haemoglobin between the two groups. The results of multiple linear regression showed that the change in haemoglobin was independent factor affecting the improvement of left ventricular mass index. Conclusions: The increase of haemoglobin was associated with improving left ventricular hypertrophy in dialysis patients. However, the beneficial effects between roxadustat and recombinant human erythropoietin on left ventricular mass index did not show clear superiority or inferiority in six months.

[Deletion of CD36 gene ameliorates glucose metabolism abnormality induced by high-fat diet and promotes liver lipid accumulation].

This study aimed to investigate the effects and the underlying mechanism of CD36 gene on glucose and lipid metabolism disorder induced by high-fat diet in mice. Wild type (WT) mice and systemic CD36 knockout (CD36-/-) mice were fed with high-fat diet for 14 weeks (n = 12). Mice were intraperitoneally injected with glucose (1 g/kg) or insulin (5 units/kg) to perform glucose tolerance test (GTT) or insulin tolerance test (ITT). Liver lipid deposition was observed by HE staining, and the contents of total triglyceride (TG), free fatty acid (FFA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were determined by automatic biochemical analyzer. Real-time PCR and Western blot were used to detect insulin signaling pathways in liver and muscle tissues of mice. The mRNA levels of genes encoding phosphoenolpyruvate carboxykinase (PEPCK) in primary hepatocytes of mice were detected by real-time PCR, and glucose detection kit was used to detect gluconeogenesis. Co-immunoprecipitation (Co-IP) and ELISA were used to detect insulin receptor ß (IRß) tyrosine phosphorylation in mouse muscle. Real-time PCR and immunofluorescence staining (IF) were used to detect the expression and location of glucose transporter 4 (GLUT4) in muscle of mice. After high-fat diet feeding, serum FFA, TG, AST and ALT levels of CD36-/- mice were significantly higher than WT mice (P < 0.05). The appearance of CD36-/- mouse liver presented fatty degeneration, and HE staining results showed increased lipid accumulation in the liver, suggesting that CD36 knockout promoted the occurrence of fatty liver. However, CD36-/- mice showed decreased fasting glucose levels, increased glucose tolerance, and decreased insulin tolerance compared with WT mice (P < 0.05), suggesting that CD36 knockout protects against the abnormal glucose metabolism induced by high-fat diet. Compared with WT mice, there was no significant difference in insulin signaling pathway in CD36-/- mouse liver, and there were no significant differences in PEPCK expression and gluconeogenesis between the two groups of primary hepatocytes. In muscle tissue, Co-IP and ELISA experiments showed that the phosphorylation level of IRß tyrosine was significantly increased in CD36-/- mice compared with that in WT mice. Besides, the levels of p-AKT in CD36-/- mouse muscle were significantly increased (P < 0.05). At the same time, IF experiment indicated that GLUT4 localization in cell membrane was enhanced in the muscle of CD36-/- mice, indicating that insulin sensitivity and glucose utilization ability were enhanced in CD36-/- mouse muscle. The results suggested that deletion of CD36 gene increased lipid accumulation in liver of mice with high-fat diet, but had no significant effect on liver gluconeogenesis. CD36 deficiency improves the abnormal glucose metabolism in mice with high-fat diet mainly through improving insulin sensitivity of muscle tissue and promoting GLUT4-mediated glucose utilization.

[Lipopolysaccharide inhibits lipophagy in HepG2 cells via activating mTOR pathway].

This study aimed to investigate the effect of lipopolysaccharide (LPS) on lipophagy in hepatocytes and the underlying mechanism. Human hepatoma cell line HepG2 was cultured in vitro, treated with 0.1 mmol/L palmitic acid (PA), and then divided into control group (0 µg/mL LPS), LPS group (10 µg/mL LPS), LPS+DMSO group and LPS+RAPA (rapamycin, 10 µmol/L) group. Lipid accumulation in hepatocytes was observed by oil red O staining. The autophagic flux of the cells was assessed using confocal laser scanning microscope after being transfected with autophagy double-labeled adenovirus (mRFP-GFP-LC3). The level of intracellular lipophagy was visualized by the colocalization of lipid droplets (BODIPY 493/503 staining) and lysosomes (lysosome marker, lysosomal associated membrane protein 1, LAMP1). The expression levels of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), ribosome protein subunit 6 kinase 1 (S6K1), p-S6K1, LC3II/I and P62 protein were examined by Western blot. The results showed that the number of red lipid droplets stained with oil red O was significantly increased in LPS group compared with that in control group (P < 0.001). Moreover, in LPS group, the number of autophagosomes was increased, while the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY were significantly decreased (P < 0.05). Meanwhile, the ratios of p-mTOR/mTOR and p-S6K1/S6K1, the ratio of LC3II/LC3I and the protein expression of P62 were significantly increased (P < 0.05) in LPS group. Furthermore, compared with LPS+DMSO group, RAPA treatment obviously reduced the number of lipid droplets and autophagosomes, and raised the number of autophagolysosomes and the colocalization rate of LAMP1 and BODIPY (P < 0.05). In conclusion, the results demonstrate that LPS inhibits lipophagy in HepG2 cells via activating mTOR signaling pathway, thereby aggravating intracellular lipid accumulation.

Gut microbiota dysbiosis-induced activation of the intrarenal renin-angiotensin system is involved in kidney injuries in rat diabetic nephropathy.

Some studies have shown that gut microbiota along with its metabolites is closely associated with diabetic mellitus (DM). In this study we explored the relationship between gut microbiota and kidney injuries of early diabetic nephropathy (DN) and its underlying mechanisms. Male SD rats were intraperitoneally injected with streptozotocin to induce DM. DM rats were orally administered compound broad-spectrum antibiotics for 8 weeks. After the rats were sacrificed, their blood, urine, feces, and renal tissues were harvested for analyses. We found that compared with the control rats, DM rats had abnormal intestinal microflora, increased plasma acetate levels, increased proteinuria, thickened glomerular basement membrane, and podocyte foot process effacement in the kidneys. Furthermore, the protein levels of angiotensin II, angiotensin-converting enzyme, and angiotensin II type 1 receptor in the kidneys of DM rats were significantly increased. Administration of broad-spectrum antibiotics in DM rats not only completely killed most intestinal microflora, but also significantly lowered the plasma acetate levels, inhibited intrarenal RAS activation, and attenuated kidney damage. Finally, we showed that plasma acetate levels were positively correlated with intrarenal angiotensin II protein expression (r = 0.969, P < 0.001). In conclusion, excessive acetate produced by disturbed gut microbiota might be involved in the kidney injuries of early DN through activating intrarenal RAS.

Caspase 3/ROCK1 pathway mediates high glucose-induced platelet microparticles shedding.

BACKGROUND: Platelet microparticles (PMPs) are closely associated with diabetic macrovascular complications. This study aimed to explore the underlying mechanisms of high glucose-induced PMPs generation. METHODS: Washed platelets, obtained from the plasma of healthy male Sprague-Dawley rats, were incubated with high glucose. PMPs were isolated using gradient centrifugation and counted by flow cytometry. Expression and activity of ROCK1 and caspase3 were evaluated by real-time PCR, Western blotting, and activity assay kit. RESULTS: High glucose enhanced PMPs shedding in the presence of collagen. The mRNA and protein levels of ROCK1, but not ROCK2, were increased in platelets incubated with high glucose. Y-27632, an inhibitor of ROCK, blocked the increased PMPs shedding induced by high glucose. Expression and activity of caspase3 were elevated in platelets under the high glucose conditions. Z-DVED-FMK, a caspase3 inhibitor, inhibited ROCK1 activity and decreased the PMPs generation under high glucose. CONCLUSION: High glucose increased PMPs shedding via caspase3-ROCK1 signal pathway.

Platelet microparticles contribute to aortic vascular endothelial injury in diabetes via the mTORC1 pathway.

Platelet microparticles (PMPs) are closely associated with diabetic macrovascular complications. The present study aimed to investigate the effects of PMPs in diabetes on aortic vascular endothelial injury and to explore the underlying mechanisms. Peritoneal injection of streptozotocin was used to generate a diabetic rat model in vivo, and human umbilical vein endothelial cells (HUVECs) treated with PMPs were used in vitro. PMP levels in the circulation and aorta tissues were time-dependently increased in streptozotocin-induced diabetic rats at weeks 4, 8, and 12 (P < 0.05). Aspirin significantly inhibited the PMP levels at each time point (P < 0.05). In diabetic rats, the endothelial nitric oxide levels were decreased significantly combined with increased endothelial permeability. PMPs were internalized by HUVECs and primarily accumulated around the nuclei. PMPs inhibited endothelial nitric oxide levels to about 50% and caused approximately twofold increase in reactive oxygen species production. Furthermore, PMPs significantly decreased the endothelial glycocalyx area and expression levels of glypican-1 and occludin (P < 0.05). Interestingly, the PMP-induced endothelial injuries were prevented by raptor siRNA and rapamycin. In conclusion, increased PMPs levels contribute to aortic vascular endothelial injuries in diabetes through activating the mTORC1 pathway.

Lipid Metabolism Disorder and Renal Fibrosis.

Since the lipid nephrotoxicity hypothesis was proposed in 1982, increasing evidence has supported the hypothesis that lipid abnormalities contributed to the progression of glomerulosclerosis. In this chapter, we will discuss the general promises of the original hypothesis, focusing especially on the role of lipids and metabolic inflammation accompanying CKD in renal fibrosis and potential new strategies of prevention.

Platelet Microparticles Mediate Glomerular Endothelial Injury in Early Diabetic Nephropathy.

BACKGROUND: Glomerular endothelium dysfunction, which plays a crucial role in the pathogenesis of early diabetic nephropathy, might be caused by circulating metabolic abnormalities. Platelet microparticles, extracellular vesicles released from activated platelets, have recently emerged as a novel regulator of vascular dysfunction. METHODS: We studied the effects of platelet microparticles on glomerular endothelial injury in early diabetic nephropathy in rats with streptozotocin-induced diabetes and primary rat glomerular endothelial cells. Isolated platelet microparticles were measured by flow cytometry. RESULTS: Plasma platelet microparticles were significantly increased in diabetic rats, an effect inhibited in aspirin-treated animals. In cultured glomerular endothelial cells, platelet microparticles induced production of reactive oxygen species, decreased nitric oxide levels, inhibited activities of endothelial nitric oxide synthase and SOD, increased permeability of the glomerular endothelium barrier, and reduced thickness of the endothelial surface layer. Conversely, inhibition of platelet microparticles in vivo by aspirin improved glomerular endothelial injury. Further analysis showed that platelet microparticles activated the mammalian target of rapamycin complex 1 (mTORC1) pathway in glomerular endothelial cells; inhibition of the mTORC1 pathway by rapamycin or raptor siRNA significantly protected against microparticle-induced glomerular endothelial injury in vivo and in vitro. Moreover, platelet microparticle-derived chemokine ligand 7 (CXCL7) contributed to glomerular endothelial injury, and antagonizing CXCL7 using CXCL7-neutralizing antibody or blocking CXCL7 receptors with a competitive inhibitor of CXCR1 and CXCR2 dramatically attenuated such injury. CONCLUSIONS: These findings demonstrate a pathogenic role of platelet microparticles in glomerular endothelium dysfunction, and suggest a potential therapeutic target, CXCL7, for treatment of early diabetic nephropathy.

Palmitic Acid-Induced Podocyte Apoptosis via the Reactive Oxygen Species-Dependent Mitochondrial Pathway.

BACKGROUND/AIMS: Chronic kidney disease (CKD) is often accompanied by hyperlipidemia, which accelerates progression of the disease. Podocyte injury can lead to dysfunction of the glomerular filtration barrier, which is associated with proteinuria, a risk marker for the progression of CKD. Our previous studies demonstrated that palmitic acid (PA) can induce podocyte apoptosis; however, the underlying mechanisms are unclear. In the present study, we investigated the specific molecular mechanisms of PA-induced apoptosis in cultured podocytes. METHODS: We cultured mouse podocytes and treated them with PA. Then, cell viability was measured using the Cell Counting Kit-8 colorimetric assay, lipid uptake was assessed by Oil Red O staining and boron-dipyrromethene staining, apoptosis was measured by flow cytometry, mitochondrial injury was assessed by JC-1 staining and transmission electron microscopy, and mitochondrial production of reactive oxygen species (ROS) was evaluated by fluorescence microscopy using the MitoSOX Red reagent. The effects of PA on the mitochondria-mediated caspase activation pathway were investigated by examining the expression of caspase-8, cleaved caspase-9, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), B-cell lymphoma 2 (Bcl-2), Bax, Bid, cytochrome c, and Fas-associated protein with death domain (FADD) using western blotting. The translocation of Bax and cytochrome c were detected by immunofluorescence. RESULTS: PA treatment significantly increased lipid accumulation and induced podocyte apoptosis. We investigated whether the two primary apoptosis signaling pathways (death receptor-mediated pathway and mitochondria-mediated pathway) were involved in the execution of PA-induced podocyte apoptosis, and found that the levels of FADD, caspase-8, and Bid did not significantly change during this process. Meanwhile, PA treatment induced an increase in Bax protein expression and a decrease in Bcl-2 protein expression, with Bax translocation to the mitochondria. Furthermore, PA treatment induced mitochondrial impairment, and triggered the release of cytochrome c from the mitochondria to cytosol, with a concomitant dose-dependent increase in the levels of cleaved caspase-9, cleaved caspase-3, and PARP. Meanwhile, PA treatment increased mitochondrial production of ROS, and the mitochondria-targeted antioxidant mitoTEMPO significantly ameliorated PA-induced podocyte apoptosis. CONCLUSION: Our findings indicated that PA induced caspase-dependent podocyte apoptosis through the mitochondrial pathway, and mitochondrial ROS production participated in this process, thus potentially contributing to podocyte injury.

Intestinal dysbiosis activates renal renin-angiotensin system contributing to incipient diabetic nephropathy.

Considerable interest nowadays has focused on gut microbiota owing to their pleiotropic roles in human health and diseases. This intestinal community can arouse a variety of activities in the host and function as "a microbial organ" by generating bioactive metabolites and participating in a series of metabolism-dependent pathways. Alternations in the composition of gut microbiota, referred to as intestinal dysbiosis, are reportedly associated with several diseases, especially diabetes mellitus and its complications. Here we focus on the relationship between gut microbiota and diabetic nephropathy (DN), as the latter is one of the major causes of chronic kidney diseases. The activation of renin angiotensin system (RAS) is a critical factor to the onset of DN, and emerging data has demonstrated a provoking and mediating role of gut microbiota for this system in the context of metabolic diseases. The purpose of the current review is to highlight some research updates about the underlying interplay between gut microbiota, their metabolites, and the development and progression of DN, along with exploring innovative approaches to targeting this intestinal community as a therapeutic perspective in clinical management of DN patients.

Liver X receptor α induces 17ß-hydroxysteroid dehydrogenase-13 expression through SREBP-1c.

Liver X receptors, including LXRα and LXRß, are known to be master regulators of liver lipid metabolism. Activation of LXRα increases hepatic lipid storage in lipid droplets (LDs). 17ß-Hydroxysteroid dehydrogenase-13 (17ß-HSD13), a recently identified liver-specific LD-associated protein, has been reported to be involved in the development of nonalcoholic fatty liver disease. However, little is known about its transcriptional regulation. In the present study, we aimed at determining whether 17ß-HSD13 gene transcription is controlled by LXRs. We found that treatment with T0901317, a nonspecific LXR agonist, increased both 17ß-HSD13 mRNA and protein levels in cultured hepatocytes. It also significantly upregulated hepatic 17ß-HSD13 expression in wild-type (WT) and LXRß-/- mice but not in LXRα-/- mice. Basal expression of 17ß-HSD13 in the livers of LXRα-/- mice was lower than that in the livers of WT and LXRß-/- mice. Moreover, induction of hepatic 17ß-HSD13 expression by T0901317 was almost completely abolished in SREBP-1c-/- mice. Bioinformatics analysis revealed a consensus sterol regulatory element (SRE)-binding site in the promoter region of the 17ß-HSD13 gene. A 17ß-HSD13 gene promoter-driven luciferase reporter and ChIP assays further confirmed that the 17ß-HSD13 gene was under direct control of SREBP-1c. Collectively, these findings demonstrate that LXRα activation induces 17ß-HSD13 expression in a SREBP-1c-dependent manner. 17ß-HSD13 may be involved in the development of LXRα-mediated fatty liver.

Inflammatory stress exacerbates the progression of cardiac fibrosis in high-fat-fed apolipoprotein E knockout mice via endothelial-mesenchymal transition.

Background Chronic inflammation plays a crucial role in the progression of cardiac fibrosis. This study investigated whether inflammation exacerbated the progression of cardiac fibrosis in high-fat-fed apolipoprotein E knockout (ApoE KO) mice via endothelial-mesenchymal transition (EndMT). Methods Twenty-four male ApoE KO mice were divided into normal chow diet (Control), high-fat diet (HFD), or high-fat diet plus 10% casein injection (inflamed) groups for 8 weeks. The body weight of ApoE KO mice was measured at each week. The lipid profile and serum amyloid A (SAA) levels were examined using clinical biochemistry and enzyme-linked immunosorbent assays, respectively. Cardiac lipid and collagen accumulation was visualised with haematoxylin-eosin (HE) and Masson's trichrome staining. EndMT-related molecule expression was examined by immunohistochemistry and Western blotting. Results SAA levels were increased in the inflamed group compared with the HFD and control groups, suggesting that inflammation was successfully induced. There were no differences in body weight among three groups at each week. Interestingly, inflammation significantly reduced serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels compared with the HFD mice. However, both foam cell formation in cardiac blood vessels and cardiac collagen deposition were increased in the inflamed group, as demonstrated by HE and Masson trichrome staining. Furthermore, inflammation reduced protein expression of CD31 and increased protein expression of alpha-smooth muscle actin (α-SMA) and collagen I, which contribute to cardiac EndMT. Conclusions Inflammatory stress exacerbates the progression of cardiac fibrosis in high-fat-fed ApoE KO mice via EndMT, suggesting that hyperlipidaemia and inflammation act synergistically to redistribute plasma lipids to cardiac tissues and accelerate the progression of cardiac fibrosis.

Interaction of RAS activation and lipid disorders accelerates the progression of glomerulosclerosis.

BACKGROUND: The activation of the renin-angiotensin system (RAS) and lipid disorders are major risk factors in progressive chronic kidney disease. This study aimed to investigate the potential synergistic mechanisms of RAS activation and lipid disorders that contribute to glomerulosclerosis. MATERIALS AND METHODS: Human renal mesangial cells (HMCs) were treated with 10(-7) mol/L angiotensin II (Ang II) or with 30 µg/ml cholesterol and 1 µg/ml 25-hydroxycholesterol (lipid loading) for 24 hours. Lipid accumulation in the cells was evaluated by Oil Red O staining and intracellular cholesterol quantitative assays. The gene and protein expression of molecules in the low-density lipoprotein receptor (LDLr) pathway, the RAS family, and the extracellular matrix were examined by real-time polymerase chain reaction and Western blotting. The translocation of sterol regulatory element-binding protein (SREBP) cleavage activating protein (SCAP), which escorts SREBP-2 from the endoplasmic reticulum (ER) to the Golgi, was examined by immunofluorescent staining. RESULTS: Ang II increased lipid droplet accumulation in HMCs. Further analysis revealed that Ang II increased the mRNA and protein expression of LDLr, SCAP, and SREBP-2. This increase was correlated with an enhanced translocation of the SCAP/SREBP-2 complex from the ER to the Golgi in HMCs that was induced by Ang II, thereby activating LDLr gene transcription. Interestingly, lipid loading increased the mRNA and protein expression of angiotensinogen, Ang II, renin, angiotensin-converting enzyme, angiotensin II type 1 receptor, and type 2 receptor in HMCs with increased mRNA and protein expression of collagen I, α-smooth muscle actin, and fibronectin. CONCLUSIONS: This study demonstrates that the interaction of RAS activation and lipid disorders accelerates the progression of glomerulosclerosis.

Activation of renin-angiotensin system is involved in dyslipidemia-mediated renal injuries in apolipoprotein E knockout mice and HK-2 cells.

BACKGROUND: Dyslipidemia and activation of renin-angiotensin system (RAS) contribute to the progression of chronic kidney disease (CKD). This study investigated possible synergistic effects of intrarenal RAS activation with hyperlipidemia in renal injuries. METHODS: Apolipoprotein knockout mice were fed with normal chow diet (control) or high fat diet (HF group) for eight weeks. Human proximal tubular epithelial cell line (HK-2) was treated without (control) or with cholesterol (30 µg/ml) plus 25-hydroxycholesterol (1 µg/ml) (lipid group) for 24 hours. The plasma lipid profile and RAS components were determined by clinical biochemistry assay and radiommunoassay, respectively. Collagen deposition in kidneys was evaluated by Masson-staining. The gene and protein expressions of molecules involved in RAS components and biomarkers of epithelial mesenchymal transition (EMT) were examined by real-time PCR, immunochemical staining, and Western blot. RESULTS: The mice fed with high-fat diet showed significant hyperlipidemia with collagen deposition in renal tubular interstitium compared to controls. The plasma levels of renin, angiotensin I, and angiotensin II were no difference in two groups. However, the kidneys of HF group showed up-regulated RAS components, which were positively associated with increased plasma levels of triglyceride, total cholesterol, and LDL. These effects were further confirmed by in vitro studies. Lipid loading induced HK-2 cells underwent EMT, which was closely associated with the increased expressions of intracellular RAS components. CONCLUSIONS: Local RAS activation was involved in hyperlipidemia-mediated renal injuries, suggesting that there are synergistic effects resulting from RAS activation with hyperlipidemia that accelerates the progression of CKD.

[Effect of inflammatory stress on hepatic cholesterol accumulation and hepatic fibrosis in C57BL/6J mice].

OBJECTIVE: To investigate whether inflammatory stress exacerbates hepatic cholesterol accumulation and liver fibrosis using a C57BL/6J mouse model of chronic inflammation. METHODS: Twelve male C57BL/6J mice were given a high-fat diet (15.0% fat, 1.25% cholesterol, 0.5% cholic acid) and randomly assigned to the normal control group (n=6; subcutaneously injected with 0.5 mL of isotonic saline, every other day for 14 weeks) or the chronic inflammation model group (n=6; subcutaneously injected with of 0.5 mL of 10% casein, every other day for 14 weeks). At the end of week 14, the animals were sacrificed and blood was collected from the left ventricle for serological analysis of inflammatory markers and lipid profile, including serum amyloid A (SAA), interleukin-6 (IL-6), total cholesterol (TC) and free cholesterol (FC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL)). Extracted liver tissues were divided for use in histological analysis (lipid accumulation and fibrosis evaluated by Oil Red O, Sirius red and Masson's trichrome staining) and quantitative fluorescence real-time PCR (to measure b-actin normalized expression of TNF-a MCP1, SREBP-2, LDLr, HMGCoA-r, ATF-6, GRP78, BMP-7, TGF-b, and collagens type I and type IV). Comparisons between groups were made by the two-samples t-test or Satterthwaite t-approximation test, collagen type I and type IV. RESULTS: Compared to the normal control group, the inflammation model group showed elevated serum IL-6 (12.55+/-4.75 vs. 32.41+/-7.42 pg/mL, P less than 0.01), reduced serum TC (14.82+/-1.56 vs. 10.62+/-0.48 mmol/L, P less than 0.01), up-regulated hepatic TNF-a mRNA expression (1.05+/-0.35 vs. 2.12+/-0.72, P less than 0.01), and elevated hepatic TC (12.10+/-2.57 vs. 23.21+/-8.75 mmol/L, P less than 0.05). In addition, the inflammation group showed abnormal lipid deposition, and increased and thickened reticular fibers. The livers of the inflammation group also showed up-regulated mRNA expression of SREBP-2 (normal control: 1.01+/-0.19 vs. 2.63+/-0.13, P less than 0.05), GRP78 (1.07+/-0.47 vs. 2.21+/-0.99, P less than 0.05), TGF-b (1.01+/-0.14 vs. 1.38+/-0.28, P less than 0.05), and collagen type I (1.02+/-0.27 vs. 1.71+/-0.51, P less than 0.05) and down-regulation of BMP-7 (1.01+/-0.15 vs. 0.55+/-0.25, P less than 0.01). CONCLUSION: Activation of the inflammatory system exacerbates hepatic cholesterol accumulation and hepatic fibrosis in C57BL/6J mice.

Activation of acetyl-CoA synthetase 2 mediates kidney injury in diabetic nephropathy.

Albuminuria and podocyte injury are the key cellular events in the progression of diabetic nephropathy (DN). Acetyl-CoA synthetase 2 (ACSS2) is a nucleocytosolic enzyme responsible for the regulation of metabolic homeostasis in mammalian cells. This study aimed to investigate the possible roles of ACSS2 in kidney injury in DN. We constructed an ACSS2-deleted mouse model to investigate the role of ACSS2 in podocyte dysfunction and kidney injury in diabetic mouse models. In vitro, podocytes were chosen and transfected with ACSS2 siRNA and ACSS2 inhibitor and treated with high glucose. We found that ACSS2 expression was significantly elevated in the podocytes of patients with DN and diabetic mice. ACSS2 upregulation promoted phenotype transformation and inflammatory cytokine expression while inhibiting podocytes' autophagy. Conversely, ACSS2 inhibition improved autophagy and alleviated podocyte injury. Furthermore, ACSS2 epigenetically activated raptor expression by histone H3K9 acetylation, promoting activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway. Pharmacological inhibition or genetic depletion of ACSS2 in the streptozotocin-induced diabetic mouse model greatly ameliorated kidney injury and podocyte dysfunction. To conclude, ACSS2 activation promoted podocyte injury in DN by raptor/mTORC1-mediated autophagy inhibition.

Outer membrane vesicles derived from gut microbiota mediate tubulointerstitial inflammation: a potential new mechanism for diabetic kidney disease.

Rationale: Chronic tubulointerstitial inflammation is a common pathological process in diabetic kidney disease (DKD). However, its underlying mechanism is largely unknown. This study aims at investigating the role of gut microbiota-derived outer membrane vesicles (OMVs) in tubulointerstitial inflammation in DKD. Methods: Gut microbiota in diabetes mellitus rats was manipulated by microbiota depletion and fecal microbiota transplantation to explore its role in tubulointerstitial inflammation. To check the direct effects of OMVs, fecal bacterial extracellular vesicles (fBEVs) were administrated to mice orally and HK-2 cells in vitro. For mechanistic investigations, HK-2 cells were treated with small interfering RNA against caspase-4 and fBEVs pre-neutralized by polymyxin B. Results: By performing gut microbiota manipulation, it was confirmed that gut microbiota mediated tubulointerstitial inflammation in DKD. In diabetic rats, gut microbiota-derived OMVs were increased and were clearly detected in distant renal tubulointerstitium. Diabetic fBEVs directly administered by gavage translocated into tubular epithelial cells and induced tubulointerstitial inflammation and kidney injury. In vitro, OMVs were internalized through various endocytic pathways and triggered cellular inflammatory response. Mechanistically, it was revealed that OMVs-derived lipopolysaccharide induced tubular inflammation, which was mediated by the activation of the caspase-11 pathway. Conclusions: Increased OMVs due to dysbiosis translocated through leaky gut barrier into distant tubulointerstitium and induced cellular inflammation and renal tubulointerstitial injury in DKD. These findings enrich the mechanism understanding of how gut microbiota and its releasing OMVs influence the development and progression of kidney disease.