Mitochondrial protein MPV17 promotes ß-cell apoptosis in diabetogenesis.

MPV17 is a mitochondrial inner membrane protein, and its deficiency can cause mitochondrial DNA (mtDNA) depletion, increase reactive oxygen species (ROS), and promote apoptosis in several cell types, suggesting that MPV17 plays a protective role in cells although the underlying mechanism remains unknown. To test whether MPV17 is also protective in diabetic kidney disease, we treated Mpv17-deficient mice with streptozotocin (STZ) and surprisingly found that they were resistant to diabetes. Mpv17 deficiency was also found to confer resistance to the diabetes induced by an insulin mutation (Ins2Akita), which represents a mouse model of monogenic diabetes characterized by proinsulin misfolding and ß-cell failure. In both STZ and Ins2Akita models, Mpv17 mutants had significantly less severe ß-cell loss and apoptosis compared with the wild-type mice. We next showed that MPV17 is expressed in ß-cells of mice normally, suggesting that MPV17 acts ß-cells autonomously to facilitate apoptosis. Consistently, Mpv17 knockdown improved the viability and ameliorated the apoptosis of cultured MIN6 cells treated with STZ and palmitic acid (PA), respectively, accompanied by prevention of caspase 3 activation. The proapoptotic effect of MPV17 in ß-cells is in contrast with its known anti-apoptotic effect in other cell types. Thus, we have identified a novel regulator of ß-cell death in diabetes development.

Regulon analysis identifies protective FXR and CREB5 in proximal tubules in early diabetic kidney disease.

Diabetic kidney disease (DKD) is the most common complication of diabetes mellitus and a leading cause of kidney failure worldwide. Despite its prevalence, the mechanisms underlying early kidney damage in DKD remain poorly understood. In this study, we used single nucleus RNA-seq to construct gene regulatory networks (GRNs) in the kidney cortex of patients with early DKD. By comparing these networks with those of healthy controls, we identify cell type-specific changes in genetic regulation associated with diabetic status. The regulon activities of FXR (NR1H4) and CREB5 were found to be upregulated in kidney proximal convoluted tubule epithelial cells (PCTs), which were validated using immunofluorescence staining in kidney biopsies from DKD patients. In vitro experiments using cultured HK2 cells showed that FXR and CREB5 protected cells from apoptosis and epithelial-mesenchymal transition. Our findings suggest that FXR and CREB5 may be promising targets for early intervention in patients with DKD.

Glucocorticoids Inhibit EGFR Signaling Activation in Podocytes in Anti-GBM Crescentic Glomerulonephritis.

Glucocorticoids are commonly used to treat anti-GBM crescentic glomerulonephritis, however, the mechanism underlying its therapeutic effectiveness is not completely understood. Since podocyte EGFR/STAT3 signaling is known to mediate the development of anti-GBM glomerulonephritis, we investigated the effect of glucocorticoids on EGFR/STAT3 signaling in podocytes. We found that the levels of phosphorylated (activated) EGFR and STAT3 in podocytes were markedly elevated in anti-GBM patients without glucocorticoids treatment, but were normalized in patients with glucocorticoids treatment. In a rat model of anti-GBM glomerulonephritis, glucocorticoids treatment significantly attenuated the proteinuria, crescent formation, parietal epithelial cell (PEC) activation and proliferation, accompanied by elimination of podocyte EGFR/STAT3 signaling activation. In cultured podocytes, glucocorticoids were found to inhibit HB-EGF-induced EGFR and STAT3 activation. The conditioned medium from podocytes treated with HB-EGF in the absence but not presence of glucocorticoids was capable of activating Notch signaling (which is known to be involved in PEC proliferation and crescent formation) and enhancing proliferative activity in primary PECs, suggesting that glucocorticoids prevent podocytes from producing secreted factors that cause PEC proliferation and crescent formation. Furthermore, we found that glucocorticoids can downregulate the expression of EGFR ligands, EGF and HB-EGF, while upregulate the expression of EGFR inhibitor, Gene 33, explaining how glucocorticoids suppress EGFR signaling. Taken together, glucocorticoids exert therapeutic effect on anti-GBM crescentic glomerulonephritis through inhibiting podocyte EGFR/STAT3 signaling and the downstream pathway that leads to PEC proliferation and crescent formation.

A novel approach to identify the mechanism of miR-145-5p toxicity to podocytes based on the essential genes targeting analysis.

MicroRNAs (miRNAs) are emerging as effective therapeutic agents. When testing whether miR-145-5p could alleviate kidney injury, we unexpectedly found that extracellular vesicles loaded with miR-145-5p induced proteinuria and podocyte foot process effacement in normal control mice. To explore the mechanism of miR-145-5p's toxicity to podocytes, we hypothesized that miR-145-5p could enter podocytes and inhibit genes essential for podocytes. We demonstrated that systemically administered miRNA can enter podocytes. Next, we predicted 611 podocyte essential genes based on single-cell RNA sequencing (RNA-seq) and found that 32 of them are predicted to be targeted by miR-145-5p. Functional annotation of the 32 podocyte essential genes revealed small GTPase-mediated signal transduction as the top pathway. We experimentally validated that miR-145-5p targeted Arhgap24 and Srgap1, the essential regulators of the Rho family of small GTPases, increased the activity of Rac1 and Cdc42, and reduced RhoA activity, accompanied by cellular injury, in podocytes. These results explain how miR-145-5p has deleterious effect on podocytes. Most importantly, our study provides a novel approach to investigate how a miRNA affects a given cell type, allowing not only identification of the molecular mechanism underlying an observed side effect of a miRNA drug but also prediction of miRNA drug toxicity on various cell types.

Cholesterol metabolic enzyme Ggpps regulates epicardium development and ventricular wall architecture integrity in mice.

During embryonic heart development, the progenitor cells in the epicardium would migrate and differentiate into noncardiomyocytes in myocardium and affect the integrity of ventricular wall, but the underlying mechanism has not been well studied. We have found that myocardium geranylgeranyl diphosphate synthase (Ggpps), a metabolic enzyme for cholesterol biosynthesis, is critical for cardiac cytoarchitecture remodelling during heart development. Here, we further reveal that epicardial Ggpps could also regulate ventricular wall architecture integrity. Epicardium-specific deletion of Ggpps before embryonic day 10.5 (E10.5) is embryonic lethal, whereas after E13.5 is survival but with defects in the epicardium and ventricular wall structure. Ggpps deficiency in the epicardium enhances the proliferation of epicardial cells and disrupts cell‒cell contact, which makes epicardial cells easier to invade into ventricular wall. Thus, the fibroblast proliferation and coronary formation in myocardium were found enhanced that might disturb the coronary vasculature remodelling and ventricular wall integrity. These processes might be associated with the activation of YAP signalling, whose nuclear distribution is blocked by Ggpps deletion. In conclusion, our findings reveal a potential link between the cholesterol metabolism and heart epicardium and myocardium development in mammals, which might provide a new view of the cause for congenital heart diseases and potential therapeutic target in pathological cardiac conditions.

Conditional loss of geranylgeranyl diphosphate synthase alleviates acute obstructive cholestatic liver injury by regulating hepatic bile acid metabolism.

Previous studies have suggested that metabolites in the mevalonate pathway are involved in hepatic bile acid metabolism, yet the details of this relationship remain unknown. In this study, we found that the hepatic farnesyl pyrophosphate (FPP) level and the ratio of FPP to geranylgeranyl pyrophosphate (GGPP) were increased in mice with acute obstructive cholestasis compared with mice that underwent a sham operation. In addition, the livers of the mice with acute obstructive cholestasis showed lower expression of geranylgeranyl diphosphate synthase (GGPPS), which synthesizes GGPP from FPP. When Ggps1 was conditionally deleted in the liver, amelioration of liver injury, as shown by downregulation of the hepatic inflammatory response and decreased hepatocellular apoptosis, was found after ligation of the common bile duct and cholecystectomy (BDLC). Subsequently, liquid chromatography/mass spectrometry analysis showed that knocking out Ggps1 decreased the levels of hepatic bile acids, including hydrophobic bile acids. Mechanistically, the disruption of Ggps1 increased the levels of hepatic FPP and its metabolite farnesol, thereby resulting in farnesoid X receptor (FXR) activation, which modulated hepatic bile acid metabolism and reduced hepatic bile acids. It was consistently indicated that digeranyl bisphosphonate, a specific inhibitor of GGPPS, and GW4064, an agonist of FXR, could also alleviate acute obstructive cholestatic liver injury in vivo. In general, GGPPS is critical for modulating acute obstructive cholestatic liver injury, and the inhibition of GGPPS ameliorates acute obstructive cholestatic liver injury by decreasing hepatic bile acids, which is possibly achieved through the activation of FXR-induced bile acid metabolism.

Reduced triglyceride accumulation due to overactivation of farnesoid X receptor signaling contributes to impaired liver regeneration following 50% hepatectomy in extra­cholestatic liver tissue.

The aim of the present study was to investigate the role of triglyceride metabolism in the effect of obstructive cholestasis on liver regeneration following 50% partial hepatectomy (PH). Obstructive cholestatic rat models were achieved via ligation of the common bile duct (BDL). Following comparisons between hepatic pathological alterations with patients with perihilar cholangiocarcinoma, rats in the 7 day post­BDL group were selected as the BDL model for subsequent experiments. Liver weight restoration, proliferating cell nuclear antigen labeling index, cytokine and growth factor expression levels, and hepatic triglyceride content were evaluated to analyze liver regeneration post­PH within BDL and control group rats. The results of the present study revealed that obstructive cholestasis impaired liver mass restoration, which occurred via inhibition of early stage hepatocyte proliferation. In addition, reduced triglyceride content and inhibited expression of fatty acid ß­oxidation­associated genes, peroxisome proliferator activated receptor α and carnitine palmitoyltransferase, were associated with an insufficient energy supply within the BDL group post­PH. Notably, the expression levels of fatty acid synthesis­associated genes, including sterol­regulatory element­binding protein­1c, acetyl­coA carboxylase 1 and fatty acid synthase were also reduced within the BDL group, which accounted for the reduced triglyceride content and fatty acid utilization. Further investigation revealed that overactivated farnesoid X receptor (FXR) signaling may inhibit fatty acid synthesis within BDL group rats. Collectively, the role of triglycerides in liver regeneration following PH in extra­cholestatic livers was identified in the present study. Additionally, the results indicated that overactivated FXR signaling­induced triglyceride reduction is associated with insufficient energy supply and therefore contributes to the extent of impairment of liver regeneration following PH within extra­cholestatic livers.

Fenofibrate decreases the bone quality by down regulating Runx2 in high-fat-diet induced Type 2 diabetes mellitus mouse model.

BACKGROUND: This study is to investigate the effect of fenofibrate on the bone quality of Type 2 diabetes mellitus (T2DM) mouse model. METHODS: T2DM mouse model was induced by high-fat-diet, and the mice were treated with fenofibrate (100 mg/kg) (DIO-FENO) or PBS (DIO-PBS) for 4 weeks. The bone microstructure and biomechanical properties of femora were analyzed by micro-CT and 3-Point bending test. The protein expression was detected by immunohistochemical staining and Western blot. The cell apoptosis was evaluated by TUNEL staining. The Bcl2, caspase 3, and osteoblast marker genes were detected by RT-qPCR. RESULTS: The biomechanical properties of bones from DIO-FENO group were significantly lower than those in the control and DIO-PBS groups. Besides, the trabecular number was lower than those of the other groups, though the cortical porosity was decreased compared with that of DIO-PBS group because of the increase of apoptotic cells. The expression of osteocalcin and collagen I were decreased after treatment with fenofibrate in T2DM mice. Moreover, the cell viability was decreased after treated with different concentrations of fenofibrate, and the expression of Runx2 decreased after treated with high dose of fenofibrate. CONCLUSION: Fenofibrate decreases the bone quality of T2DM mice through decreasing the expression of collagen I and osteocalcin, which may be resulted from the down regulation of Runx2 expression.

Zoledronic acid, an FPPS inhibitor, ameliorates liver steatosis through inhibiting hepatic de novo lipogenesis.

Currently, there is no standard therapy for non-alcoholic fatty liver disease (NAFLD), and statins have been developed as a first-line pharmaceutical therapeutic option for NAFLD-associated dyslipidemia. However, prolonged statins therapy has side effects, as statins inhibit HMG-CoA reductase, an enzyme at the very beginning of the mevalonate pathway. Here, we found that zoledronic acid (ZA), an inhibitor of farnesyl diphosphate synthase in the downstream mevalonate pathway, could attenuate hepatic lipid accumulation and improve liver injury in both high-fat diet-induced C57BL/6J mice and ob/ob mice. Moreover, the hepatic lipid metabolism was largely inhibited after ZA administration in high-fat diet-induced obese mice. Mechanically, ZA inhibited SREBP-1c-mediated de novo lipogenesis through suppressing RhoA activation via decreasing farnesyl diphosphate and geranylgeranyl diphosphate levels. In conclusion, our data provide a novel application of ZA in improving hepatic steatosis.

Geranylgeranyl Diphosphate Synthase Modulates Fetal Lung Branching Morphogenesis Possibly through Controlling K-Ras Prenylation.

G proteins play essential roles in regulating fetal lung development, and any defects in their expression or function (eg, activation or posttranslational modification) can lead to lung developmental malformation. Geranylgeranyl diphosphate synthase (GGPPS) can modulate protein prenylation that is required for protein membrane-anchoring and activation. Here, we report that GGPPS regulates fetal lung branching morphogenesis possibly through controlling K-Ras prenylation during fetal lung development. GGPPS was continuously expressed in lung epithelium throughout whole fetal lung development. Specific deletion of geranylgeranyl diphosphate synthase 1 (Ggps1) in lung epithelium during fetal lung development resulted in neonatal respiratory distress syndrome-like disease. The knockout mice died at postnatal day 1 of respiratory failure, and the lungs showed compensatory pneumonectasis, pulmonary atelectasis, and hyaline membranes. Subsequently, we proved that lung malformations in Ggps1-deficient mice resulted from the failure of fetal lung branching morphogenesis. Further investigation revealed Ggps1 deletion blocked K-Ras geranylgeranylation and extracellular signal-related kinase 1 or 2/mitogen-activated protein kinase signaling, which in turn disturbed fibroblast growth factor 10 regulation on fetal lung branching morphogenesis. Collectively, our data suggest that GGPPS is essential for maintaining fetal lung branching morphogenesis, which is possibly through regulating K-Ras prenylation.