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Renal proximal tubular angiotensinogen (AGT) is increased by hyperglycemia (HG) in diabetes mellitus, which augments intrarenal angiotensin II formation, contributing to the development of hypertension and kidney injury. Sodium-glucose cotransporter 2 (SGLT2) is abundantly expressed in proximal tubular cells (PTCs). The present study investigated the effects of canagliflozin (CANA), a SGLT2 inhibitor, on HG-induced AGT elevation in cultured PTCs. Mouse PTCs were treated with 5-25 mM glucose. CANA (0-10 œM) was applied 1 h before glucose treatment. Glucose (10 mM) increased AGT mRNA and protein levels at 12 h (3.06 ± 0.48-fold in protein), and 1 and 10 œM CANA as well as SGLT2 shRNA attenuated the AGT augmentation. CANA did not suppress the elevated AGT levels induced by 25 mM glucose. Increased AGT expression induced by treatment with pyruvate, a glucose metabolite that does not require SGLT2 for uptake, was not attenuated by CANA. In HG-treated PTCs, intracellular reactive oxygen species levels were elevated compared with baseline (4.24 ± 0.23-fold), and these were also inhibited by CANA. Furthermore, tempol, an antioxidant, attenuated AGT upregulation in HGtreated PTCs. HG-induced AGT upregulation was not inhibited by an angiotensin II receptor antagonist, indicating that HG stimulates AGT expression in an angiotensin II-independent manner. These results indicate that enhanced glucose entry via SGLT2 into PTCs elevates intracellular reactive oxygen species generation by stimulation of glycolysis and consequent AGT augmentation. SGLT2 blockade limits HG-induced AGT stimulation, thus reducing the development of kidney injury in diabetes mellitus. [ABSTRACT FROM AUTHOR]

To better understand the role of the inward-rectifying K channel Kir4.1 (KCNJ10) in the distal nephron, we initially studied a global Kir4.1 knockout mouse (gKO), which demonstrated the hypokalemia and hypomagnesemia seen in SeSAME/EAST syndrome and was associated with reduced Na/Cl cotransporter (NCC) expression. Lethality by ~3 wk, however, limits the usefulness of this model, so we developed a kidney-specific Kir4.1 "knockdown" mouse (ksKD) using a cadherin 16 promoter and Cre-loxP methodology. These mice appeared normal and survived to adulthood. Kir4.1 protein expression was decreased ~50% vs. wild-type (WT) mice by immunoblotting, and immunofluorescence showed moderately reduced Kir4.1 staining in distal convoluted tubule that was minimal or absent in connecting tubule and cortical collecting duct. Under control conditions, the ksKD mice showed metabolic alkalosis and relative hypercalcemia but were normokalemic and mildly hypermagnesemic despite decreased NCC expression. In addition, the mice had a severe urinary concentrating defect associated with hypernatremia, enlarged kidneys with tubulocystic dilations, and reduced aquaporin-3 expression. On a K/Mg-free diet for 1 wk, however, ksKD mice showed marked hypokalemia (serum K: 1.5 ± 0.1 vs. 3.0 ± 0.1 mEq/l for WT), which was associated with renal K wasting (transtubular K gradient: 11.4 ± 0.8 vs. 1.6 ± 0.4 in WT). Phosphorylated-NCC expression increased in WT but not ksKD mice on the K/Mg-free diet, suggesting that loss of NCC adaptation underlies the hypokalemia. In conclusion, even modest reduction in Kir4.1 expression results in impaired K conservation, which appears to be mediated by reduced expression of activated NCC. [ABSTRACT FROM AUTHOR]

R4128 --> 732.9 --> Introduction: The Ca2+‐activated Cl‐ channel Anoctamin‐1 (Ano1) regulates multiple cell functions including cell proliferation, survival, and migration. We previously reported that overexpression of Ano1 is associated with hyperproliferation of pulmonary artery endothelial cells isolated from patients with idiopathic pulmonary arterial hypertension. We also showed that Ano1 is expressed not only in the plasma membrane (PM), but also in the mitochondria. However, the physiological and pathological roles of mitochondrial Ano1 have not been fully investigated. Aim: To investigate the role of mitochondria‐localized Ano1 on the regulation of mitochondrial dynamics and reactive oxygen species (ROS) generation. Methods: Stable overexpression and knockdown of Ano1 were employed in HEK293T cells and mouse embryonic fibroblasts (MEFs), respectively. Cells were used for biochemical and cell biological assays. Results: First, mitochondrial localization of Ano1 in addition to the PM was confirmed by live cell imaging of HEK293T cells expressing GFP‐tagged Ano1 and immunostaining of endogenous Ano1 in MEFs. Next, a co‐immunoprecipitation assay showed that overexpressed Ano1 in HEK293T cells was associated with optic atrophy 1 (OPA1) which is an inner mitochondrial membrane (IMM) protein and a critical regulator of mitochondrial fusion, cristae formation, and bioenergetics. We also confirmed the interaction between endogenous Ano1 and OPA1 using wild‐type and OPA1‐knockout MEFs. Overexpression of wild‐type Ano1 in HEK293T cells facilitated proliferation, but a pore‐dead mutant (Ano1‐R621E) did not. Importantly, Ano1 overexpressing HEK293T cells have higher cell death rates in response to oxidative stress compared to control cells. To further understand the role of endogenous Ano1 expression on mitochondrial functions, we next employed MEFs stably overexpressing shRNA targeting Ano1. Quantifying mitochondrial morphology from live MEFs expressing mitochondria‐matrix targeted GFP revealed that Ano1‐knockdown (Ano1‐KD) MEFs contained more elongated mitochondria compared to cells stably expressing control shRNA. Importantly, we also found higher expression of mitofusin 2 and long‐form OPA1 in Ano1‐KD MEFs compared to control cells. The expression levels of other fission/fusion proteins were not altered by Ano1‐KD. Lastly, significantly lower levels of mitochondrial superoxide in Ano1‐KD cells and a tendency towards lower cellular oxidative levels in Ano1‐KD cells compared to controls were observed by live cell imaging with mitochondrial superoxide‐sensitive dye MitoSOX Red and protein carbonyl quantification in the whole cell lysates, respectively. Summary: Ano1, expressed in both the IMM and the PM, is involved in the maintenance of mitochondrial morphology and ROS, possibly by modulating fusion protein expression levels. Future studies will include the development of genetically engineered mitochondria‐targeted Ano1 to more precisely dissect the roles of IMM‐localized Ano1 in mitochondrial functions. [ABSTRACT FROM AUTHOR]

R4274 --> Anoctamin‐1 (Ano1) forms a Ca2+‐activated chloride channel found at the plasma membrane (PM) of several different cell types, including endothelial cells (ECs). We previously showed Ano1 overexpression in pulmonary artery ECs isolated from patients with idiopathic pulmonary arterial hypertension (PAH), which may be associated with hyperproliferative phenotype of pulmonary artery ECs in PAH. However, the detailed molecular mechanisms explaining how Ano1 modulates proliferative signaling are still not clear. In addition, recent proteomics studies showed that Ano1 may interact with inner mitochondrial membrane (IMM) proteins in addition to plasma membrane (PM)‐localized proteins. However, the expression levels of Ano1 and its functions in other cellular compartments including mitochondria are not fully understood. Here, we report that Ano1 is expressed in the mitochondria in pulmonary artery ECs and may be involved in EC proliferation by interacting with an IMM protein, optic atrophy 1 (OPA1) which is a critical regulator of mitochondrial bioenergetics and cristae formation. First, mitochondrial localization of Ano1 in addition to PM was found by protein fractionation and immunostaining of Ano1 in rat lung microvascular ECs (RLMVECs). We also confirmed mitochondrial Ano1 expression by live cell imaging of HEK293T cells expressing GFP‐tagged Ano1. Next, a co‐immunoprecipitation assay showed that overexpressed Ano1 interacts with OPA1 in HEK293T cells, indicating that Ano1 is expressed at IMM. We also confirmed that endogenous Ano1 interacts with OPA1 in mouse embryonic fibroblasts. Overexpression of Ano1 in HEK293T cells showed higher proliferation rate compared to controls (cells stably expressing GFP) at rest, but showed higher cell death rates in response to oxidative stress compared to control compared to control cells. Lastly, we found that treatment of a small molecule Ano1 activator Eact promotes apoptosis in pulmonary artery ECs from patients with idiopathic PAH, which likely occurs via depolarization of mitochondrial membrane potential by opening of mitochondrial Ano1. In summary, we found that 1) Ano1 is expressed in both the IMM and the PM, 2) Ano1 and OPA1 physically interact at IMM, 3) overexpression of Ano1 does increase proliferation, but Ano1 activation promotes cellular apoptosis. Interactions between Ano1 and OPA1 may provide a molecular mechanism underlying the hyperproliferation of pulmonary artery ECs during PAH. In addition, Ano1 activation may serve as a new therapeutic tool in PAH associated with hyperproliferative ECs. [ABSTRACT FROM AUTHOR]

Mitochondrial fragmentation frequently occurs in chronic pathological conditions as seen in various human diseases. In fact, abnormal mitochondrial morphology and mitochondrial dysfunction are hallmarks of heart failure (HF) in both human patients and HF animal models. A link between mitochondrial fragmentation and cardiac pathologies has been widely proposed, but the physiological relevance of mitochondrial fission and fusion in the heart is still unclear. Recent studies have increasingly shown that posttranslational modifications (PTMs) of fission and fusion proteins are capable of directly modulating the stability, localization, and/or activity of these proteins. These PTMs include phosphorylation, acetylation, ubiquitination, conjugation of small ubiquitin-like modifier proteins, O -linked- N -acetyl-glucosamine glycosylation, and proteolysis. Thus, understanding the PTMs of fission and fusion proteins may allow us to understand the complexities that determine the balance of mitochondrial fission and fusion as well as mitochondrial function in various cell types and organs including cardiomyocytes and the heart. In this review, we summarize present knowledge regarding the function and regulation of mitochondrial fission and fusion in cardiomyocytes, specifically focusing on the PTMs of each mitochondrial fission/fusion protein. We also discuss the molecular mechanisms underlying abnormal mitochondrial morphology in HF and their contributions to the development of cardiac diseases, highlighting the crucial roles of PTMs of mitochondrial fission and fusion proteins. Finally, we discuss the future potential of manipulating PTMs of fission and fusion proteins as a therapeutic strategy for preventing and/or treating HF.