Targeted Disruption of a Proximal Tubule-Specific TMEM174 Gene in Mice Causes Hyperphosphatemia and Vascular Calcification.

BACKGROUND: The proximal tubules play a critical role in phosphate (Pi) homeostasis by reabsorbing Pi via sodium-dependent Pi cotransporters. NPT2A is a major proximal-specific Pi cotransporter, whose expression is regulated by circulating hormones, such as parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). In this study, we aimed to find a novel regulator in Pi homeostasis. METHODS: Using RNA-seq and RT-qPCR analysis, we identified proximal tubule cell-enriched genes. We next used RNAi screening of the identified proximal tubular cell-enriched genes to identify a novel proximal tubule-specific gene that contributes to FGF23- and PTH-mediated inhibition of Pi uptake and NPT2 reduction. We created mice lacking this novel regulator of Pi homeostasis to examine whether the novel regulator contributes to Pi homeostasis in vivo. RESULTS: We identified 54 kidney-enriched genes, 19 of which are expressed in renal primary proximal tubule cells. One of the proximal tubule-specific genes, TMEM174, interacted with NPT2A, and its knockdown blocked the reduction of NPT2A protein by FGF23 and PTH treatments in human and opossum proximal tubule cells. TMEM174 KO mice had significantly increased levels of serum Pi, FGF23, and PTH, resulting in vascular calcification. CONCLUSIONS: TMEM174 is a novel regulator of Pi homeostasis that interacts with NPT2A.

The CDK9-cyclin T1 complex mediates saturated fatty acid-induced vascular calcification by inducing expression of the transcription factor CHOP.

Vascular calcification (or mineralization) is a common complication of chronic kidney disease (CKD) and is closely associated with increased mortality and morbidity rates. We recently reported that activation of the activating transcription factor 4 (ATF4) pathway through the saturated fatty acid (SFA)-induced endoplasmic reticulum (ER) stress response plays a causative role in CKD-associated vascular calcification. Here, using mouse models of CKD, we 1) studied the contribution of the proapoptotic transcription factor CCAAT enhancer-binding protein homologous protein (CHOP) to CKD-dependent medial calcification, and 2) we identified an additional regulator of ER stress-mediated CHOP expression. Transgenic mice having smooth muscle cell (SMC)-specific CHOP expression developed severe vascular apoptosis and medial calcification under CKD. Screening of a protein kinase inhibitor library identified 16 compounds, including seven cyclin-dependent kinase (CDK) inhibitors, that significantly suppressed CHOP induction during ER stress. Moreover, selective CDK9 inhibitors and CRISPR/Cas9-mediated CDK9 reduction blocked SFA-mediated induction of CHOP expression, whereas inhibitors of other CDK isoforms did not. Cyclin T1 knockout inhibited SFA-mediated induction of CHOP and mineralization, whereas deletion of cyclin T2 and cyclin K promoted CHOP expression levels and mineralization. Of note, the CDK9-cyclin T1 complex directly phosphorylated and activated ATF4. These results demonstrate that the CDK9-cyclin T1 and CDK9-cyclin T2/K complexes have opposing roles in CHOP expression and CKD-induced vascular calcification. They further reveal that the CDK9-cyclin T1 complex mediates vascular calcification through CHOP induction and phosphorylation-mediated ATF4 activation.

Simultaneous inhibition of FXR and TGR5 exacerbates atherosclerotic formation.

Simultaneous activation of bile acid receptors farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5) by INT-767 significantly reduces atherosclerotic formation. In this study, we investigated the effect of simultaneous inactivation of these bile acid receptors in atherosclerosis and which bile acid receptor mediates the anti-atherogenic effect of INT-767. To investigate the role of simultaneous inactivation of FXR and TGR5 in vivo, we generated LDL receptor knockout (LDLR) KO mice with FXR and TGR5 dual deficiency, which exhibited severe atherosclerosis and aortic inflammation through nuclear factor κΒ activation. The lipid-lowering effects of INT-767 were completely blocked by FXR single deficiency but not TGR5 single deficiency. INT-767 was able to block atherosclerotic formation and decrease levels of aortic cytokines and chemokines in LDLR KO mice under either FXR or TGR5 single deficiency. Dual deficiency of FXR and TGR5 completely blocked the anti-atherogenic and anti-inflammatory effects of INT-767 in LDLR KO mice. We demonstrated that 1) FXR and TGR5 dual deficiency exacerbated the development of atherosclerosis and 2) the anti-atherogenic effect of INT-767 requires the anti-inflammatory effect but not the lipid-lowering effect through the simultaneous activation of FXR and TGR5. Our results indicate that dual activation of FXR and TGR5 is a promising strategy for treating atherosclerosis.

Lipidomic insight into cardiovascular diseases.

Cardiovascular disease is a primary cause of mortality worldwide. Therefore, it is of major interest to identify sensitive molecular markers that predict cardiovascular events and point to therapeutic strategies that will increase lifespans. Dysregulated lipid metabolism is recognized as an established risk factor in cardiovascular diseases. However, it is still largely unknown which specific lipid molecular species reflect cardiovascular risk. In addition, understanding the whole lipidome signature in vascular pathophysiology is challenging. Recent advancements of mass-spectrometry allow researchers to detect each individual lipid species from unbiased small samples. In this review, we update the current research on lipidomic approaches in cardiovascular diseases.

Activation of the IKK2/NF-κB pathway in VSMCs inhibits calcified vascular stiffness in CKD.

IKK2/NF-κB pathway-mediated inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2/NF-κB pathway in medial calcification remains to be elucidated. In this study, we found that chronic kidney disease (CKD) induces inflammatory pathways through the local activation of the IKK2/NF-κB pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2/NF-κB pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NF-κB by SMC-specific IκBα deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2/NF-κB pathway induced cell death of VSMCs by reducing anti-cell death gene expression, whereas activation of NF-κB reduced CKD-dependent vascular cell death. In addition, increased calcification of extracellular vesicles through the inhibition of the IKK2/NF-κB pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death in vitro and in vivo. This study reveals that activation of the IKK2/NF-κB pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles.

Activation of the IKK2-NFκB pathway in VSMCs inhibits calcified vascular stiffness in CKD by reducing the secretion of calcifying extracellular vesicles.

IKK2-NFκB pathway mediated-inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2-NFκB pathway in medial calcification remains to be elucidated. In this study, we found that CKD induces inflammatory pathways through the local activation of the IKK2-NFκB pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2-NFκB pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NFκB by SMC-specific IκB deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2-NFκB pathway induced apoptosis of VSMCs by reducing anti-apoptotic gene expression, whereas activation of NFκB reduced CKD-dependent vascular cell death. In addition, increased calcifying extracellular vesicles through the inhibition of the IKK2-NFκB pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death. This study reveals that activation of the IKK2-NFκB pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles.

MEF2D-NR4A1-FAM134B2-mediated reticulophagy contributes to amino acid homeostasis.

ABBREVIATIONS: AAD: amino acid deficiency; APOC3: apolipoprotein C3; BACH1: BTB domain and CNC homolog 1; CEBP: CCAAT enhancer binding protein; DDIT3/CHOP: DNA damage inducible transcript 3; EBSS: Earle's Balanced Salt Solution; EIF2AK4/GCN2: eukaryotic translation initiation factor 2 alpha kinase 4; ER: endoplasmic reticulum; HisOH: histidinol; ISR: integrated stress response; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF2D: myocyte enhancer factor 2D; MTOR: mechanistic target of rapamycin kinase; NR4A1: nuclear receptor subfamily 4 group A member 1; RETREG1/FAM134B: reticulophagy regulator 1; RTN2: reticulon 2, TF: transcription factor; TFEB: transcription factor EB; ZBTB10: zinc finger and BTB domain containing 10.

Free Deoxycholic Acid Exacerbates Vascular Calcification in CKD through ER Stress-Mediated ATF4 Activation.

BACKGROUND: Our metabolome approach found that levels of circulating, free deoxycholic acid (DCA) is associated with the severity of vascular calcification in patients with CKD. However, it is not known whether DCA directly causes vascular calcification in CKD. METHODS: Using various chemicals and animal and cell culture models, we investigated whether the modulation of DCA levels influences vascular calcification in CKD. RESULTS: CKD increased levels of DCA in mice and humans by decreasing urinary DCA excretion. Treatment of cultured VSMCs with DCA but no other bile acids (BAs) induced vascular calcification and osteogenic differentiation through endoplasmic reticulum (ER) stress-mediated activating transcription factor-4 (ATF4) activation. Treatment of mice with Farnesoid X receptor (FXR)-specific agonists selectively reduced levels of circulating cholic acid-derived BAs, such as DCA, protecting from CKD-dependent medial calcification and atherosclerotic calcification. Reciprocal FXR deficiency and DCA treatment induced vascular calcification by increasing levels of circulating DCA and activating the ER stress response. CONCLUSIONS: This study demonstrates that DCA plays a causative role in regulating CKD-dependent vascular diseases through ER stress-mediated ATF4 activation.

GPAT4-Generated Saturated LPAs Induce Lipotoxicity through Inhibition of Autophagy by Abnormal Formation of Omegasomes.

Excessive levels of saturated fatty acids are toxic to vascular smooth muscle cells (VSMCs). We previously reported that mice lacking VSMC-stearoyl-CoA desaturase (SCD), a major enzyme catalyzing the detoxification of saturated fatty acids, develop severe vascular calcification from the massive accumulation of lipid metabolites containing saturated fatty acids. However, the mechanism by which SCD deficiency causes vascular calcification is not completely understood. Here, we demonstrate that saturated fatty acids significantly inhibit autophagic flux in VSMCs, contributing to vascular calcification and apoptosis. Mechanistically, saturated fatty acids are accumulated as saturated lysophosphatidic acids (LPAs) (i.e. 1-stearoyl-LPA) possibly synthesized through the reaction of GPAT4 at the contact site between omegasomes and the MAM. The accumulation of saturated LPAs at the contact site causes abnormal formation of omegasomes, resulting in accumulation of autophagosomal precursor isolation membranes, leading to inhibition of autophagic flux. Thus, saturated LPAs are major metabolites mediating autophagy inhibition and vascular calcification.

An N-terminal-truncated isoform of FAM134B (FAM134B-2) regulates starvation-induced hepatic selective ER-phagy.

Autophagy is a conserved system that adapts to nutrient starvation, after which proteins and organelles are degraded to recycle amino acids in response to starvation. Recently, the ER was added to the list of targets of autophagic degradation. Autophagic degradation pathways of bulk ER and the specific proteins sorted through the ER are considered key mechanisms in maintaining ER homeostasis. Four ER-resident proteins (FAM134B, CCPG1, SEC62, and RTN3) have been identified as ER-resident cargo receptors, which contain LC3-interacting regions. In this study, we identified an N-terminal-truncated isoform of FAM134B (FAM134B-2) that contributes to starvation-induced ER-related autophagy. Hepatic FAM134B-2 but not full-length FAM134B (FAM134B-1) is expressed in a fed state. Starvation drastically induces FAM134B-2 but no other ER-resident cargo receptors through transcriptional activation by C/EBPß. C/EBPß overexpression increases FAM134B-2 recruitment into autophagosomes and lysosomal degradation. FAM134B-2 regulates lysosomal degradation of ER-retained secretory proteins such as ApoCIII. This study demonstrates that the C/EBPß-FAM134B-2 axis regulates starvation-induced selective ER-phagy.

Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells.

Emerging evidence indicates that upregulation of the ER stress-induced pro-osteogenic transcription factor ATF4 plays an important role in vascular calcification, a common complication in patients with aging, diabetes, and chronic kidney disease (CKD). In this study, we demonstrated the pathophysiological role of ATF4 in vascular calcification using global Atf4 KO, smooth muscle cell-specific (SMC-specific) Atf4 KO, and transgenic (TG) mouse models. Reduced expression of ATF4 in global ATF4-haplodeficient and SMC-specific Atf4 KO mice reduced medial and atherosclerotic calcification under normal kidney and CKD conditions. In contrast, increased expression of ATF4 in SMC-specific Atf4 TG mice caused severe medial and atherosclerotic calcification. We further demonstrated that ATF4 transcriptionally upregulates the expression of type III sodium-dependent phosphate cotransporters (PiT1 and PiT2) by interacting with C/EBPß. These results demonstrate that the ER stress effector ATF4 plays a critical role in the pathogenesis of vascular calcification through increased phosphate uptake in vascular SMCs.

Saturated phosphatidic acids mediate saturated fatty acid-induced vascular calcification and lipotoxicity.

Recent evidence indicates that saturated fatty acid-induced (SFA-induced) lipotoxicity contributes to the pathogenesis of cardiovascular and metabolic diseases; however, the molecular mechanisms that underlie SFA-induced lipotoxicity remain unclear. Here, we have shown that repression of stearoyl-CoA desaturase (SCD) enzymes, which regulate the intracellular balance of SFAs and unsaturated FAs, and the subsequent accumulation of SFAs in vascular smooth muscle cells (VSMCs), are characteristic events in the development of vascular calcification. We evaluated whether SMC-specific inhibition of SCD and the resulting SFA accumulation plays a causative role in the pathogenesis of vascular calcification and generated mice with SMC-specific deletion of both Scd1 and Scd2. Mice lacking both SCD1 and SCD2 in SMCs displayed severe vascular calcification with increased ER stress. Moreover, we employed shRNA library screening and radiolabeling approaches, as well as in vitro and in vivo lipidomic analysis, and determined that fully saturated phosphatidic acids such as 1,2-distearoyl-PA (18:0/18:0-PA) mediate SFA-induced lipotoxicity and vascular calcification. Together, these results identify a key lipogenic pathway in SMCs that mediates vascular calcification.

Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.

Bile acid signaling is a critical regulator of glucose and energy metabolism, mainly through the nuclear receptor FXR and the G protein-coupled receptor TGR. The purpose of the present study was to investigate whether dual activation of FXR and TGR5 plays a significant role in the prevention of atherosclerosis progression. To evaluate the effects of bile acid signaling in atherogenesis, ApoE-/- mice and LDLR-/- mice were treated with an FXR/TGR5 dual agonist (INT-767). INT-767 treatment drastically reduced serum cholesterol levels. INT-767 treatment significantly reduced atherosclerotic plaque formation in both ApoE-/- and LDLR-/- mice. INT-767 decreased the expression of pro-inflammatory cytokines and chemokines in the aortas of ApoE-/- mice through the inactivation of NF-κB. In addition, J774 macrophages treated with INT-767 had significantly lower levels of active NF-κB, resulting in cytokine production in response to LPS through a PKA dependent mechanism. This study demonstrates that concurrent activation of FXR and TGR5 attenuates atherosclerosis by reducing both circulating lipids and inflammation.

Endoplasmic reticulum stress effector CCAAT/enhancer-binding protein homologous protein (CHOP) regulates chronic kidney disease-induced vascular calcification.

BACKGROUND: Cardiovascular diseases such as atherosclerosis and vascular calcification are a major cause of death in patients with chronic kidney disease (CKD). Recently, the long-awaited results of the Study of Heart and Renal Protection trial were reported. This large randomized clinical trial found that an extensive cholesterol-lowering therapy through the combination of simvastatin and ezetimibe significantly reduced cardiovascular diseases in a wide range of patients with CKD. However, the mechanism by which this cholesterol-lowering therapy reduces CKD-dependent vascular diseases remains elusive. The objective of the present study was to determine the contribution of the oxysterol-induced pro-apoptotic transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP) on the pathogenesis of CKD-dependent cardiovascular diseases through endoplasmic reticulum stress signaling. METHODS AND RESULTS: CKD increased levels of serum oxysterols such as 7-ketocholesterol in human patients and ApoE(-/-) mice. Treatment with simvastatin plus ezetimibe strongly reduced levels of serum oxysterols and attenuated CKD-dependent atherosclerosis, vascular cell death, vascular calcification, and cardiac dysfunction. This therapy also reduced aortic endoplasmic reticulum stress induced by CKD. The short hairpin RNA-mediated knockdown of CHOP and activating transcription factor-4 in vascular smooth muscle cells attenuated oxysterol-induced mineralization, osteogenic differentiation, and endoplasmic reticulum stress. In addition, CHOP deficiency protected ApoE(-/-) mice from CKD-dependent vascular calcification, cardiac dysfunction, and vascular cell death. CONCLUSIONS: These data reveal that the cholesterol-lowering therapy of simvastatin plus ezetimibe attenuates CKD-dependent vascular diseases through a reduction of oxysterol-mediated endoplasmic reticulum stress. CHOP plays a crucial role in the pathogenesis of CKD-dependent vascular calcification.