bluuebunny/crossref_metadata_2025 huggingface dataset:
Heme Oxygenase‐1 Attenuates the Production of Endothelin‐1 from Human Glomerular Endothelial Cells Induced by Serum from Placental Ischemic Hypertensive Rats
Authors: Bakrania Bhavisha , Spradley Frank T. , Satchell Simon , Stec David E. , Granger Joey P.
Published: November 2023

Preeclampsia is a disorder associated with maternal hypertension, endothelial dysfunction and reductions in renal hemodynamics. Placental ischemia leads to increases in circulating maternal anti‐angiogenic and pro‐inflammatory cytokines that induce endothelin‐1 (ET‐1), a potent vasoconstrictor. Placental ischemia‐induced hypertension in rats is not only attenuated by blocking the ET‐1 system, but also by inducing heme oxygenase‐1 (HO‐1), which catabolizes heme to yield carbon monoxide, biliverdin and bilirubin. Although it is known that systemic induction of HO‐1 reduces renal ET‐1 content with placental ischemia‐induced hypertension, it is unknown whether this pathway has a direct effect to inhibit renal glomerular endothelial ET‐1 production induced by circulating placental ischemic factors. We tested the hypothesis that HO‐1 and its metabolites attenuate ET‐1 production induced by serum from placental ischemic (RUPP; reduced uterine perfusion pressure) versus normal pregnant (NP) rats in conditionally immortalized human glomerular endothelial cells. Cells were cultured; starved for 48 h; and treated for 12 h resulting in the following 6 groups having N=6/group: 1) 5% NP serum, 2) 5% RUPP serum, 3) RUPP serum + cobalt protoporphyrin (CoPP, 25μM), 4) RUPP serum + CormA1 (100μM, CO‐releasing compound), 5) RUPP serum + biliverdin (10μM) and 6) RUPP serum + bilirubin (10μM). Mean arterial pressure in RUPP animals was significantly increased compared to the NP group (RUPP, 120±5.3 mmHg vs. NP, 99±1.9 mmHg, P=0.007). ET‐1 production was greater in cells treated with RUPP versus NP serum (RUPP, 178.3±7.3 pg/mL vs. NP, 68.1±4.1 pg/mL, P<0.001). HO‐1 induction abolished (RUPP + CoPP, 0.79±0.11 pg/mL, P<0.001) ET‐1 production, whereas its metabolite bilirubin reduced ET‐1 release (RUPP + bilirubin, 153.7±2.1 pg/mL P=0.007). In conclusion, the results of this study demonstrate that circulating factors released during the development of placental ischemia‐induced hypertension target the maternal glomerular endothelium to increase pro‐hypertensive ET‐1 and this effect can be inhibited by HO‐1 and its metabolites.
Inhibition of hypoxia inducible factor‐1α in the renal medulla contributes to fructose‐induced salt‐sensitive hypertension
Authors: Hu Junping , Zhu Qing , Li Pin‐Lan , Boini Krishna M. , Li Ningjun
Published: November 2023

Excessive fructose ingestion has been closely associated with hypertension and shown to increase the salt sensitivity of blood pressure. We have previously demonstrated that high salt‐induced activation of hypoxia inducible factor (HIF)‐1α in the renal medulla is an important adaptive mechanism to remove extra sodium load and maintain normal blood pressure in response to a high salt intake. The present study tested the hypothesis that dietary fructose inhibits high salt‐induced activation of HIF‐1α in the renal medulla and thereby increases salt‐sensitivity of blood pressure. Adult Sprague‐Dawley rats were divided into 4 groups: low salt diet (LS, 0.4%NaCl), high salt diet (HS, 8% NaCl), LS+fructose (20% in drinking water) and HS+fructose. After 10 day treatment, mean arterial pressure (MAP) was significantly higher in rats fed with HS+fructose than all other groups, whereas MAP was not different among groups of LS, HS and LS+fructose (MAP: 103 ± 4.7 mmHg in LS group, 104 ± 2.4 in LS+fructose, 105 ± 3.4 in HS, and 120 ± 2.03 in HS+fructose, P<0.05). Interestingly, high salt‐induced increase of renal medullary HIF‐1α protein levels was significantly inhibited by fructose. The relative levels of HIF‐1α in the renal medulla in groups of LS, HS, LS+Fructose and HS+Fructose were 1 ± 0.10, 2.05 ± 0.36, 0.18 ± 0.008, 0.19 ± 0.05 (P<0.05), respectively. The mRNA levels of HIF‐1α target gene heme oxygenase (HO)‐1 was also inhibited by 69% in HS+Fructose group compared with HS group. Furthermore, overexpression HIF‐1α transgene in the renal medulla corrected the reduced levels of HIF‐1α and its target gene HO‐1 in the renal medulla and attenuated the increase of blood pressure induced by HS+fructose (MAP: 104 ± 3.9 mmHg in LS group, 107 ± 2.3 in LS+fructose, 107 ± 3.1 in HS, 124 ± 3.4 in HS+fructose, and 114 ± 1.4 in HS+fructose+HIF‐1α transgene, P<0.05). These results suggested that inhibition of HIF‐1α‐mediated gene activation in the renal medulla participates in the mechanisms of fructose‐induced salt‐dependent hypertension, and manipulating HIF‐1α signaling pathway may be used as a therapeutic strategy in fructose‐induced salt‐dependent hypertension.

Support or Funding Information

support: NIH grant: HL89563 and HL106042
Sources for anti‐inflammatory annexin A1 signals during acute anti‐Thy‐1.1 nephritis
Authors: Labes Robert , Dittert Philipp , Bachmann Sebastian , Paliege Alexander
Published: November 2023

Leukocyte infiltration presents a hallmark feature of immune‐mediated acute kidney injury and defective resolution of inflammation may promote progression towards chronic kidney failure. Leukocytes exert distinctive effects during this process and may either aggravate kidney damage or foster renal repair. The glucocorticoid‐inducible protein Annexin A1 has been shown to induce resolution of inflammation and to shift macrophage polarization towards the anti‐inflammatory and repair‐promoting M2 phenotype. The cellular source of the putative Annexin A1 signal and its regulation during the course of renal inflammation remains to be elucidated.

Adult Wistar rats were injected with an antibody directed against the Thy‐1.1 protein located on mesangial cells to induce mesangioproliferative glomerulonephritis. Animals were examined after 24h (initiation phase), 5d (proliferation phase), and 15d (resolution phase). Regulation of Annexin A1 was studied by qPCR and immunohistochemistry. Annexin A1+ cells were characterized by triple labelling immunofluorescence using antibodies for CD68 (monocytes and macrophages), CD206 (macrophages with M2 polarization), CD3, CD4, and CD8 (T‐cell subpopulations), CD20 (B‐lymphocytes), and myeloperoxidase (neutrophil granulocytes). Quantification of immunoreactive cells was performed by cell counting on confocal micrographs.

Renal Annexin A1 mRNA levels increased rapidly after induction of anti‐Thy‐1.1 nephritis (24h: +92±16%; 5d: +128±19%; 15d: +78±33% relative to controls; p<.05). Immunofluorescence labelling showed an interstitial accumulation of total, and CD206+ M2 macrophages during the proliferation and resolution phase (5d: +150±40% and +260±29%; 15d: +378±43% and +600±100% relative to controls; p<.05). Elevated numbers of CD68+/CD206+/Annexin A1+ interstitial M2 macrophages were revealed by triple labelling studies at d5 and d15 (+240±27% and +450±80% relative to controls; p<.05). Abundant expression of Annexin A1 was further found in CD4+ and CD8+ T‐lymphocytes but their number remained unaffected by renal inflammation. CD20+ B‐lymphocytes were negative for Annexin A1. Granulocytes were found in subset of glomeruli at 24h and d5. All granulocytes stained positive for Annexin A1.

In conclusion, we have shown a significant increase of Annexin A1 gene expression and protein abundance during the course of anti‐Thy‐1.1 nephritis. The increased amount of Annexin A1+ leukocytes in animals with anti‐Thy‐1.1 nephritis suggest that these cells present a major source for anti‐inflammatory and pro‐resolving signals during renal inflammation.

Support or Funding Information

Deutsche Forschungsgemeinschaft; FOR1368
Uncoupling Protein‐2 Deficiency Protects Kidney Function in Ageing Mice
Authors: Carvalho Carla , Schiffer Tomas , Hansell Peter , Palm Fredrik
Published: November 2023

Increased kidney oxygen consumption due to increased mitochondrial leak respiration contributes to chronic kidney disease. Physiological leak respiration in the kidney is mainly mediated by uncoupling protein‐2 (UCP‐2) since it is the only UCP expressed in this tissue. However, it is not known how UCP‐2 affects long term kidney function in the ageing kidney although we hypothesize that UCP‐2 deficiency protects kidney function in aged mice.

In this study, 2 year old wild‐type and UCP‐2 deficient mice (B6.129S.UCP2lJ, Jackson Lab) were placed in metabolic cages for 24h urine collection followed by determination of conscious arterial blood pressure using the tail cuff method and glomerular filtration rate using a single bolus injection of FITC‐inulin.

UCP‐2 deficient mice had similar water and food intake as well as body and kidney weights compared to wild‐type mice. However, systolic blood pressure was higher in UCP‐2 deficient mice (systolic 95±5 versus 88±3 mmHg) whereas diastolic pressure was similar in the two groups (62±4 versus 61±2 mmHg). Interestingly, urinary protein excretion was approximately 30% lower in UCP‐2 deficient mice and glomerular filtration rate significantly higher (307±30 versus 228±30 μl/min) compared to corresponding wild‐type animals.

In conclusion, these results demonstrate that UCP‐2 deficiency protects long‐term kidney function in ageing mice by reducing urinary protein leakage and maintaining normal glomerular filtration rate. Thus, treatment options aiming to reduce the age‐related activation of UCP‐2 may improve kidney status in the ever growing population of older people.

Support or Funding Information

Swedish Research Council and Swedish Diabetes Foundation
Three‐layered proteomic characterization of a novel ACTN4 mutation reveals its pathogenic potential in FSGS
Authors: Rinschen Markus M , Bartram Malte , Benzing Thomas , Beck Bodo B
Published: November 2023

Genetic diseases constitute the most important cause for end‐stage renal disease in children and adolescents. Mutations in the ACTN4 gene encoding the actin‐binding protein alpha‐actinin‐4, are a rare cause of autosomal dominant familial focal segmental glomerulosclerosis (FSGS). Here, we report the identification of a novel, disease‐causing ACTN4 mutation (p.G195D de novo) in a sporadic case of childhood FSGS using next generation sequencing. Proteome analysis by quantitative mass spectrometry of patient derived urinary epithelial cells indicated that ACTN4 levels were significantly decreased compared to healthy controls. By resolving the peptide bearing the residue aspartic acid on position 195 (p.195D) we could verify that the mutant protein is less abundantly expressed as compared to the wildtype protein. Further analyses revealed that the decreased stability of p.G195D is associated with increased ubiquitylation in the vicinity of the mutation site. We next defined the ACTN4 interactome which was predominantly composed of cytoskeletal modulators and LIM domain containing proteins. Interestingly, this entire group of proteins, including several highly specific ACTN4 interactors, was globally decreased in the patient derived cell clones. Taken together, these data suggest a mechanistic link between ACTN4 instability and proteome perturbations observed in the ACTN4 interactome. Our findings advance the understanding of dominant effects exerted by ACTN4 mutations in FSGS. This study illustrates the potential of genomics and complementary, high‐resolution proteomics analyses for the assesment of novel, potentially causative genetic variants in the context of rare disorders.

Support or Funding Information

DFG
Role of Vav2 in Podocyte Inflammasome Activation and Glomerular Injury during Hyperhomocysteinemia
Authors: Conley Sabena M. , Chen Zhida , Xia Min , Boini Krishna M. , Gehr Todd W. , Li Pin‐Lan
Published: November 2023

Recently, our lab has reported that Vav2, a member of the guanine nucleotide exchange factor (GEF) family contributes to the activation of NADPH oxidase (NOX) in a membrane lipid signaling platform in response to elevated levels of homocysteine (Hcys). However, it remains unknown whether Vav2‐mediated NOX activation is able to trigger NLRP3 inflammasomes in podocytes and thereby lead to podocyte dysfunction and glomerular injury associated with hyperhomocysteinemia (hHcys). The present study was designed to answer this question by using pharmalogical interventions in vitro and in vivo. In our experiments, murine podocytes were pretreated with either a Rac‐1 inhibitor, NSC23766 or Vav2 activator, uridine triphosphate (UTP) and then stimulated with Hcys for 24 hours. Confocal microscopic analysis showed that treatment with UTP increased the colocalization of inflammasome proteins NLRP3 with ASC or with caspase‐1, suggesting NLRP3 inflammasome formation. However, pretreatment of podocytes with NSC23766 blocked Hcys‐induced inflammasome formation. Similar to Hcys, Vav2 activator, UTP increased caspase‐1 activation and consequent IL‐1β production in podocytes, signifying NLRP3 inflammasome activation. However, attenuated caspase‐1 activation and lower IL‐1β levels were observed when podocytes were treated with Vav2 inhibitor, NSC23766. Furthermore, Hcys and UTP treatment resulted in increased levels of GTP‐bound Rac‐1 in comparison to NSC23766 treated podocytes. In our in vivo studies using mice fed a folate‐free (FF) diet to induce hHcys, we found that these mice had increased inflammasome formation, enhanced desmin expression, elevated urinary protein or albumin, and decreased podocin expression in glomeruli. These hHcys‐induced changes were substantially attenuated in mice receiving NSC23766. Our results suggest that Vav2 is a key signaling molecule in mediating Hcys‐induced podocyte inflammasome formation and activation and consequent podocyte dysfunction and glomerular injury.

Support or Funding Information

Supported by NIH grants DK054927, HL07356 & HL057244.
Characterization of the Electrogenic Secretory Response in the Porcine Colon following in vivo Challenge with Brachyspira hyodysenteriae and “Brachyspira hampsonii”
Authors: Enns Cole B , Harding John C , Loewen Matthew E
Published: November 2023

The effects of Brachyspira hyodysenteriae and “Brachyspira hampsonii” infections on the agonist induced electrogenic secretion were assessed to determine their contribution to the pathophysiology of swine dysentery. Proximal, apex and distal sections of colon were compared in healthy and diseased purebred Yorkshire grower pigs in Ussing chambers. Activation of secretion via isoproterenol (adrenergic agonist), carbachol (cholinergic agonist) and forskolin / 3‐isobutyl‐1‐methylxanthine (IBMX) demonstrated a significantly decreased change in short‐circuit current (Isc) in infected pigs in all three sections of colon compared to control, confirming the previous observation that swine dysentery is not secretory in nature. Tissue resistances did not account for this difference, rather, it was attributed to a decrease in Cl− secretion as indicated by a significant decrease in bumentanide inhibitable short‐circuit current in infected animals. qRT‐PCR was conducted to determine if a decrease in gene transcript was responsible for the loss of Cl− secretory function. The major chloride transport proteins of the epithelium, such as the Cystic Fibrosis transmembrane conductance regulator (CFTR), Transmembrane member protein 16A (TMEM16A) and Na‐K‐Cl co‐transporter 1 (NKCC1) were compared between healthy and diseased animals in all three sections of colon. Inflammatory mediators were also assed to probe potential mechanism behind the transporter modulation. mRNA expression for these channels were determined to be elevated in the infected samples compared to controls. This suggests that these Brachyspira species reduce the Cl− secretory response by directly inhibiting Cl− channels or by inhibiting the translation of mRNA into protein.

Support or Funding Information

Natural Sciences and Engineering Research Council (NSERC) 371364 / 2010 to MEL & Alberta Livestock and Meat Association (ALMA) 2013R054R to JCH, MEL & JEH
The toxin of pseudomonas aeruginosa pyocyanin induces lysosomal degradation of CFTR: Consequences for strategies to correct ΔF508‐CFTR
Authors: Cormet‐Boyaka Estelle , Chang Sheng‐Wei , Balsiger Robert , Boyaka Prosper
Published: November 2023

CFTR plays a key role in maintenance of lung fluid homeostasis. Mutations in the cftr gene resulting in defects in CFTR protein and/or function lead to cystic fibrosis (CF), a genetic disease associated with chronic infection and inflammation. Pseudomonas aeruginosa (P. aeruginosa) is the major pathogen in the lung of CF patients. One of the toxins produced by P. aeruginosa is pyocyanin (PCN), a redox‐active metabolite. PCN was shown to impair CFTR chloride transport but the mechanism is still unclear. We have evidence that PCN decreases CFTR expression by inducing its lysosomal degradation in human bronchial epithelial cells. Since we recently reported that activation of the ERK signaling pathway by cigarette smoke negatively regulates CFTR expression, we investigated whether PCN impairs CFTR expression via activation of the ERK MAPK pathway. Our results show that PCN induces activation of the ERK pathway in human bronchial epithelial cells. Inhibition of the ERK MAPK pathway using specific inhibitors prevented the PCN‐induced reduction of CFTR protein. Conversely, inhibition of the JNK or P38 MAPK pathways had no protective effect. Pharmacological compounds are being developed to rescue the processing defect of the most common CF‐causing mutant, ΔF508‐CFTR. We hypothesized that PCN, which is abundantly present in the lung of CF patients, will hamper the beneficial effect of CFTR correctors. CFBE cells, a human bronchial cell line derived from a CF patient, were treated with the corrector VX‐661 which allowed rescue of ΔF508‐CFTR as observed by detection of mature (Band C) CFTR. Addition of PCN resulted in the loss of rescued‐CFTR. Interestingly, presence of MEK/ERK inhibitor was able to prevent decrease of mature CFTR induced by PCN. Our results indicate that PCN present in the lung of CF patients could hamper correction of ΔF508‐CFTR and identify the MEK/ERK pathway as a new molecular target.

Support or Funding Information

NIH
Exosome‐Associated Iron Release during Respiratory Virus Co‐Infection Enhances Pseudomonas aeruginosa Biofilm Growth
Authors: Bomberger Jennifer , Lashua Lauren , Fischer Douglas , Hendricks Matthew
Published: November 2023

Clinical observations link respiratory virus infection and colonization with the opportunistic pathogen Pseudomonas aeruginosa in Cystic Fibrosis (CF) patients, but the mechanism underlying this interaction is not understood. The transition of P. aeruginosa from an acute to chronic infection often involves the development of highly antibiotic resistant biofilm communities in the lung, which contribute to disease progression in patients. We hypothesized that respiratory viral co‐infection promotes P. aeruginosa biofilm growth. Using a novel live‐cell imaging model, we observed that in the presence of respiratory syncytial virus (RSV) co‐infection, P. aeruginosa biofilm formation on airway epithelial cells (AECs) is significantly increased. The innate immune response to virus infection, measured by type III interferon (IFN‐λ) production, peaks at the same time as virus‐induced biofilm growth, and interestingly, treatment of AECs with IFN‐λ replicates the enhanced biofilm formation observed during virus co‐infection. The biofilm stimulatory activity was isolated to purified exosomes in conditioned media obtained from apical membrane of AECs infected with RSV. Conditioned media from RSV‐infected AECs was observed to have significantly increased levels of iron, and iron levels were also elevated in bronchoalveolar lavage fluid obtained from mice infected with RSV, suggesting that RSV infection disrupts host iron homeostasis in the airway epithelium. Iron is an essential nutrient for P. aeruginosa growth and biofilm maturation, and chelation of iron inhibits biofilm growth on human AECs. Although virus infection did not change the abundance of iron transporters in AECs, RSV did enhance apical secretion of exosome‐associated transferrin, a host iron‐binding protein previously shown to promote P. aeruginosa biofilm growth. Depleting transferrin from conditioned media blocked the biofilm stimulatory effect of RSV co‐infection. We are currently investigating the mechanism by which interferon signaling induces transferrin packaging and secretion in exosomes to stimulate P. aeruginosa biofilm growth. The antiviral interferon response to respiratory viral infection enhances susceptibility to secondary bacterial infections, and our data suggest a novel mechanism by which these pathways contribute to the development of chronic pulmonary P. aeruginosa infections. Moreover, these studies increase our understanding of how the host contributes to pathogen persistence in the airway.

Support or Funding Information

This work was supported by NIH K99/R00HL098342, P30DK072506, R56HL123771 and CFF Research Grant (JMB).
Interleukin‐6 Transactivation of the Mineralocorticoid Receptor
Authors: Wynne Brandi Michele , Kronk Trinity Augusta , Al‐Khalili Otor , Mallick Rickta , Ko Benjamin , Eaton Douglas C , Hoover Robert S
Published: November 2023

Hypertension is characterized by increased sodium (Na+) reabsorption along the aldosterone‐sensitive distal nephron (ASDN) as well as a chronic systemic inflammation. Interleukin‐6 (IL‐6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na+ reabsorption within the ASDN does not always correlate with increases in aldosterone (Aldo), the primary hormone which modulates Na+ reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased ASDN Na+ reabsorption may occur independent of Aldo stimulation is critical. We hypothesize that IL‐6 can transactivate the MR, increasing Na+ reabsorption via reactive oxygen species (ROS) generation and activation of the sodium‐chloride cotransporter (NCC). We transfected MR‐EGFP tagged constructs into a model of murine distal convoluted tubule (mDCT15) cells. Cells were then treated with vehicle, Aldo (100–250nM) or IL‐6 (100ng/mL), fixed and visualized with confocal microscopy. MR‐EGFP expression was observed in the cytosol in the vehicle treated cells. However, following IL‐6 treatment (30 min), MR‐EGFP fluorescence was observed translocated into the nucleus, similar to Aldo‐treated positive controls. To confirm activation of downstream transcription factors, mDCT15 cells were transfected with mineralocorticoid response element (MRE)‐luciferase reporter constructs, treated with vehicle, Aldo (100nM, 24hr) or IL‐6 (100ng/mL, 24hr) and luciferase assays performed (48hrs). With IL‐6 treatment, luciferase activity was increased approximately 1.5 fold over MRE‐only transfected cells. Excessive MR stimulation is known to produce ROS generation. Thus, we determined if ROS generation stimulates increased NCC‐mediated Na+ uptake. Using mDCT15 cell monolayers, thiazide‐sensitive 22Na+‐uptake studies were performed. Increased 22Na+ uptake was observed after ROS generation (pyrogallol, 50μM) as compared to vehicle (1834±19 vs. 1506±18nmol/mg, p<0.0001, n=6), which was then reduced with the addition of the superoxide dismutase mimetic tempol (pyrogallol+tempol, 250μM) (1618±27nmol/mg vs. pyrogallol+tempol, p<0.0001, n=6). These data suggest that: IL‐6 can transactivate the MR leading to nuclear translocation and subsequent MRE activation, and ROS generation increases ASDN Na+ reabsorption via NCC providing evidence for alternate mechanisms of ASDN Na+ uptake during conditions where Aldo‐mediated MR stimulation may not occur.

Support or Funding Information

NIDDK085097‐RSH2T32DK7656‐21 Emory Renal Division‐BMW
Identification of Paraoxonase‐2 as a Novel Modulator of the Epithelial Na+ Channel
Authors: Shi Shujie , Buck Teresa M. , Kinlough Carol L. , Marciszyn Allison L. , Chalfie Martin , Brodsky Jeffrey L. , Hughey Rebecca P. , Kleyman Thomas R.
Published: November 2023

Paraoxonase‐2 (PON‐2) is a membrane‐bound lactonase with unique anti‐oxidative and anti‐atherosclerotic properties, and thus has been associated with cardiovascular diseases. PON‐2 shares key structural elements with MEC‐6, an essential component of the C. elegans mechanosensitive ion channel complex required for the worm's touch response. At the core of this channel complex, MEC‐6 interacts with two pore‐forming subunits, MEC‐4 and MEC‐10 and enhances channel activity. MEC‐4 and MEC‐10 belong to the same family as the epithelial Na+ channel (ENaC), a key mediator of Na+ uptake in the aldosterone sensitive distal nephron (ASDN). We therefore examined PON‐2 expression in the ASDN, and immunofluorescence microscopy indicated tubular‐specific expression of PON‐2 that included aquaporin 2‐expressing cells. When PON‐2 was co‐expressed with individual ENaC subunits in HEK293 cells, PON‐2 and each of the three ENaC subunits could be co‐immunoprecipitated. In contrast, PON‐2 co‐immunoprecipitated only the α subunit of ENaC when all three subunits were expressed. When PON‐2 was co‐expressed with ENaC in Xenopus oocytes, both whole cell Na+ currents and ENaC surface expression were reduced. This inhibitory effect was ENaC‐specific, as PON‐2 had no effect on ROMK activity. However, the PON‐2 inhibitory effect was lost with a PON‐2 mutant (D268A) that abrogates the lactonase activity of PON‐2, likely as a result of interfering with calcium binding. Together, our results indicate that PON‐2 interacts with ENaC and modulates channel expression.
Tyrosine Phosphorylation of Mitochondrial Ca2+ Uniporter Dictates Mitochondrial Ca2+ Overload and Cardiomyocyte Death
Authors: Mishra Jyotsna , Hurst Stephen , Jhun Bong Sook , Sheu Shey‐Shing , O‐Uchi Jin
Published: November 2023

Introduction

Mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) is critical for the cardiomyocyte survival and death. Basal tyrosine phosphorylation (P‐Y) of MCU was reported from mass spectroscopy of human tissues. However, it is still unclear whether the post‐translational modifications of the MCU (e.g. phosphorylation) modulates its channel properties as well as mitochondrial and cellular functions in cardiomyocytes.

Hypothesis

P‐Y of MCU activates mitochondrial Ca2+ uptake and induces mitochondrial Ca2+ overload and cardiomyocyte death.

Methods

Non‐phospho‐mimetic MCUs (MCU‐YFs; changed from Y to F) were generated by PCR‐based site direct mutagenesis. Mitochondrial Ca2+ concentration was measured by matrix‐targeted Ca2+ biosensors using confocal microscopy.

Results

a1‐adrenoceptor (a1‐AR) signaling activated mitochondrial matrix‐localized proline‐rich tyrosine kinase 2 (Pyk2) and enhanced the interaction between Pyk2 and MCU, which subsequently increased P‐Y of MCU and mitochondrial Ca2+ uptake in rat cardiomyocytes. We confirmed that two tyrosine sites showed an increase of phosphorylation levels in response to a1‐AR stimulation in situ (Fig. A). In addition, the overexpression of these MCU‐YFs failed to increase mitochondrial Ca2+ uptake in response to cytosolic Ca2+ elevation, whereas wild‐type MCU overexpression dramatically accelerated mitochondrial Ca2+ uptake compared to control cells (Fig. B). Moreover, persistent a1‐AR stimulation increased mitochondrial reactive oxygen species (mROS) generation, cytochrome C release, and cardiomyocyte death. These effects were abolished by the overexpression of a dominant‐negative mutant of MCU in cardiomyocytes.

Conclusion

MCU contains Pyk2‐specific phosphorylation site(s) and P‐Y of MCU activates its channel function. Persistent P‐Y of MCU induces mitochondrial Ca2+ overload, mROS generation and apoptosis in cardiomyocytes.

Support or Funding Information

This work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).
The Physiological Effect of Altering Mitochondrial Calcium Uptake in the Renal Cortical Collecting Duct
Authors: Galarza‐Paez Laura I , Eaton Douglas C , Zou Li , Al‐Khalili Otor , Ma Heping , Sands Jeff M , Klein Janet D , Thai Tiffany L
Published: November 2023

Principal cells in the renal collecting duct (CCD) regulate total body salt and water. The principal cells respond differently to extracellular signals depending upon whether the signals are delivered to the apical or basolateral surface of the cells. Specifically, basolateral ATP increases epithelial sodium channel (ENaC) activity and transepithelial sodium reabsorption whereas apical ATP is inhibitory. Paradoxically, both of these opposing signaling pathways are mediated by an increase intracellular calcium ([Ca2+]i). We have shown recently that mitochondria localize beneath the apical and basolateral plasma membranes in principal cells to reduce the rate of [Ca2+]i diffusion away from the poles of the cell and, thereby, allow elevated [Ca2+]i within specific regions of the cell. Ca2+ uptake into mitochondria is a two‐step process, occurring via the voltage dependent anion channel (VDAC) in the outer mitochondrial membrane, a channel which can transport anions or cations depending on conformation, and then through the mitochondrial calcium uniporter in the mitochondrial inner membrane. After Ca2+ is rapidly sequestered by the mitochondria, it is only slowly released, promoting [Ca2+]i buffering. We asked whether genetically inducing a deficiency in mitochondrial Ca2+ uptake that would prevent the development of locally high [Ca2+]i would produce a renal or cardiovascular phenotype due to Ca2+‐dependent misregulation of ENaC. We compared blood pressure and kidney morphology in wild type mice and those lacking VDAC3 at baseline and after two weeks of 8% salt diet. At baseline, kidney morphology and blood pressure were similar between the two strains (106.3±4.85 mmHg vs 94.8±3.87 mmHg, P>0.1). After two weeks of high salt diet, VDAC3 KO mice showed glomerular atrophy that was not seen in wild type animals and they had a higher blood pressure than their wild type littermates (127.5mmHg ±6.89 vs 109.0mmHg ±3.77, P<0.05). These data suggest that a genetic defect in mitochondrial Ca2+ uptake results in the development of renal and cardiovascular damage in response to high salt.

Support or Funding Information

NIH 5R25DK101390, NIH R37‐DK037963 to DCE, NIH R01‐DK100582 to HM and AHA 13POST16820072 to TLT.
Loss of ZnT2‐mediated Zinc Transport Leads to Cytoplasmic Zinc Accumulation and Impairs Mammary Gland Involution
Authors: Rivera Olivia C , Hennigar Stephen R , Kelleher Shannon L
Published: November 2023

The zinc (Zn) transporter ZnT2 is expressed in highly specialized exocrine tissues and imports Zn into mitochondria and vesicles. ZnT2‐mediated Zn transport is particularly critical in the secreting mammary gland (MG). In addition to importing Zn into secretory vesicles for secretion into milk, we recently reported ZnT2‐null mice (ZnT2ko) have impaired MG differentiation and secretion, which leads to reduced milk volume and sub‐optimal milk composition. Moreover, re‐localization of ZnT2 from secretory vesicles to lysosomes by TNF‐α stimulates lysosomal Zn import and initiates lysosomal mediated cell death, thus activating MG remodeling and involution. We hypothesized that loss of ZnT2 function would lead to impaired execution of mechanisms that drive MG remodeling. This may be particularly relevant to breast health, as non‐synonymous genetic variation in ZnT2 that impairs ZnT2 function is common and insufficient MG remodeling may predispose women to breast cancer. To determine the effects of the loss of ZnT2 function on involution we used ZnT2ko mice and their wild‐type (WT) littermates. Whole mount analysis revealed the MG of ZnT2ko mice, forced to undergo involution by removal of their offspring, more closely resembled a lactating MG than that of involuting MGs from WT mice. The MG area repopulated by adipocytes increased by 12% in ZnT2ko mice compared to a 37% increase in WT mice (P<0.001). To determine if the loss of ZnT2 impaired Zn sequestration, Zinpyr‐1, a cell permeable fluorescent probe was used to image labile Zn. In WT mice, labile Zn accumulation was only observed in numerous extruded apoptotic cells. In contrast, in ZnT2ko mice fewer apoptotic cells were noted, and cytoplasmic Zn accumulation was observed in the non‐apoptotic mammary epithelial cells, which was associated with reduced Stat3 activation (P<0.05). Furthermore, macrophage infiltration, a late event that occurs during MG involution, was reduced by 83% in ZnT2ko mice (P<0.05) and macrophages in ZnT2ko mice displayed a circular morphology, compared to the irregular morphology observed in WT mice, suggesting impaired macrophage activation. Our results indicate that the loss of ZnT2 leads to cytoplasmic Zn accumulation, which inhibits activation of key signals initiating or potentiating the cellular events characterizing involution.

Support or Funding Information

Intramural funds to SLK
Role of the Low‐voltage Activated Ca2+ Current (LVA) in the Vestibular Afferent Neurons in Culture
Authors: Galván Maricruz Rangel , Vega y Saenz de Miera María Rosario , Eguibar Enrique Soto
Published: November 2023

Vestibular‐afferent neurons (VANs) resting electrical discharge may vary from regular to irregular action potential intervals spanning a continuum of variation coefficients. The specific firing properties of each neuron are correlated with the soma size and the synaptic input, however, firing patterns of these neurons cannot be explained completely by means of those characteristics. It has been proposed that intrinsic properties (variable expression and density of the ion channels) contribute to establish the firing pattern observed in VANs. It has been found that the after‐hyperpolarization phase (AHP) of the action potential (AP) is an essential component determining the firing regularity of VANs. The AHP involve the activation of the hyperpolarization‐activated current (Ih), Ca2+‐activated K+ current (IKCa), Na+‐activated K+ current (IKNa), low‐voltage activated K+ current (IKLV) and low‐voltage activated Ca2+ current (LVA). Based on previous results it has been suggested that heterogeneous distribution of the LVA currents contribute to differentiate the discharge pattern of VANs. Here, we study the functional role of LVA current on VANs‐discharge. Using whole‐cell patch‐clamp recording, we observed a blocking effect of Nickel (300 μM) and Mibefradil (2 μM) on LVA currents. Current‐clamp recordings showed that Ni2+ modified the morphology of action potentials (AP), decreasing the amplitude and increasing the duration of the AP, on the other hand Mibefradil decreased amplitude and threshold (T) of the AP. Moreover, the frequency of the repetitive firing generated by a long depolarization pulse (1.5 × T) was decreased after applying both Ni2+ and Mibefradil blockers. Discharge regularity is measured by a coefficient of variation (CV) of interspike intervals obtained with a Gaussian white noise stimulation protocol. LVA current blocked with Mibefradil produced a trend toward regular firing. Sinusoidal stimuli shown a group of cells in which the AP number increased and another group where the AP number decreased. Our results showed that VANs possess a LVA current and that their heterogeneous expression would contribute to explain some of the differences in the discharge pattern in these neurons.

Support or Funding Information

Dr. Enrique Soto EguibarInstitute of Physiology ‐ BUAP A.P. 406Puebla, Pue. Postal code: 72570 MexicoTel: (52‐222) 2295500 est 7316Fax: (52‐222) 2295500 ext 7301
Lipopolysaccharide Induces TLR4 Dependent Activation of VRAC (LRRC8) in Nodose Neurons and May thereby Impair Satiety Signal
Authors: Wang Runping , Zhang Yanhui , Cicha Michael Z , Sah Rajan , Chapleau Mark W , Abboud Francois M
Published: November 2023

Recent reports indicate that lipopolysaccharide (LPS) reduces the response of vagal afferent nodose neurons to CCK and leptin and thereby reduces satiety signaling and contributes to hyperphagia (de La Serre et. al. 2015). Here we defined the specific ion channel involved in the response of nodose neurons to LPS. We suspected that a Chloride (Cl−) channel may be responsible based on our earlier finding that high fat diet reduces the CCK‐sensitive Ca2+‐dependent Cl− current in intestinal vagal afferent nodose neurons (FASEB J 2015. 29: 818.4). We used whole‐cell voltage‐clamp on dissociated nodose neurons from C57BL/6 mice, and found that application of 10μg/ml LPS induced a large, slowly developing inward Cl− current in 14 of 18 cells (17.2±3.0 pA/pF, n=14). The current was attenuated (4.0±1.1 pA/pF, n=14; p<0.01) when intracellular Cl− concentration was reduced from 133mM to 4mM. We concluded that this LPS‐induced Cl− channel is the ubiquitous Volume‐Regulated Anion Channel (VRAC) with Leucine Rich Repeats Containing protein 8a (LRRC8a or SWELL1) for two reasons: 1) the current was significantly reduced (6.6±2.0 pA/pF, n=6, p<0.01) by DCPIB (4‐(2‐butyl‐6,7‐dichloro‐2‐cyclopentylindan‐1‐on‐5‐yl)oxybutyric acid), a selective blocker of VRAC; and 2) the current was reduced from 18.1±2.9 pA/pF (n=7) to 4.3±1.3 pA/pF in neurons exposed to LRRC8a‐shRNA (n=5, p<0.01) compared to a scrambled shRNA control. Our data also suggests that the effect of LPS on the channel is not a direct one but is mediated by TLR4 (toll‐like receptor 4) since HEK293 cells, which express SWELL1/LRRC8a but not TLR4, rarely responded to LPS (1 out of 6 cells), and the response was not significant (7.8±4.7 pA/pF, n=6, p>0.05) compared to the positive control response to hypoosmolarity (30.9±7.9 pA/pF, n=5, p<0.01). We conclude that LPS activates LRRC8 in nodose neurons. Excessive TLR4 mediated activation of this channel by LPS may inhibit the CCK and leptin induced satiety signal, leading to hyperphagia in high fat diet and LPS induced obesity.

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HL014388‐43
Possible role of myosin 2 and myosin 5C in the anterograde trafficking of KCa3.1 in a polarized epithelium
Authors: Farquhar Rachel , Rodrigues Ely , Hamilton Kirk L.
Published: November 2023

Recently, we reported that KCa3.1 is trafficked to the basolateral membrane (BLM) of epithelial cells by a Rab1‐ and Rab8‐dependent mechanism (Bertuccio et al. PlosONE 9:e92013, 2014). Here, we extend our work on the role of microtubule/microfilament function in the trafficking of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We used our FRT epithelial cell line stably expressing KCa3.1‐BLAP/Bir‐A‐KDEL (cells grown on filters), immunoblot and Ussing chamber experiments in this study. Our immunoblot approach was to streptavidin label channels that arrive at the BLM to determine the effects of inhibitors of microtubule/microfilament function on the trafficking of the KCa3.1. We examined the effect colchicine, a microtubule inhibitor (10 μM for 0, 3 and 5 hr); ML‐9 an inhibitor of myosin light chain kinase (10 μM for 0, 3, and 5 hr) and 2,3‐butanedione monoxime (BDM) an inhibitor of muscle and non‐muscle myosin 2 and myosin 5 (10 mM for 0, 3, 5 hr) on trafficking of KCa3.1 to the BLM. As determined by immunoblot, colchicine significantly reduced trafficking of KCa3.1 by 70±8% (at 5 hr, P≤0.05, n=5); ML‐9 decreased trafficking of the channel by 70±3% (5 hr, n=5) and BDM reduced trafficking of the channel by 47±11% (5 hr, P≤0.05, n=5) compared to controls. We examined the effects of the inhibitors on the functional expression of KCa3.1 by Ussing chamber experiments. By using 1‐EBIO (100 μM, an activator of KCa3.1) to stimulate current via KCa3.1, we determined that colchicine (5 hr treatment) decreased 1‐EBIO‐stimulated current by 40±20% (P≤0.05, n=5); ML‐9 (5 hr) reduced stimulated current by 60±10% (P≤0.05, n=5) and BDM (5 hr) decreased stimulated current by 46±8% (P≤0.05, n=5) compared the controls. These data suggest that myosin 2 and myosin 5 may be critical for the trafficking of KCa3.1 to BLM of polarized epithelial cells.

Support or Funding Information

This work was supported by UORG and Dean's Fund grants from the Univ. of Otago (KLH).
Identification of ANO1 (TMEM16A) activator purified from Cucumis sativus: Its role for treatment of cystic fibrosis and gastrointestinal disorders
Authors: Kazi Mirajul Hoque , Saha Tultul , Woodward Owen M , Guggino William B
Published: November 2023

BACKGROUND

CFTR functions as a chloride (Cl−) channel. Its dysfunction limits Cl− secretion leading to viscous mucus in the airway and intestinal lumen. Increases in cytosolic Ca2+ activate the epithelial Calcium activate Chloride Channel (CaCC), which provides an alternative Cl− secretory pathway in CF. In the present study, we purified a positive fraction from Cucumis sativus and isolated compounds that activated ANO1, member of the CaCC family.

METHODS

The crushed fruit extracted was collected as aqueous solution by a fruit crusher followed by fractionation using High Performance Liquid Chromatography (HPLC). Short‐circuit current (Isc) and chloride (Cl−) conductance were measured in T84 cells by Ussing chamber. ANO1 current was measured in xenopus expression system.

RESULTS

Administration of crude as well as purified compound to the apical side of epithelial cells activates Isc in intact as well as basolaterally permeabilized colonic T84 cells in a dose dependent manner. The stimulated Cl− secretion can be inhibited by DIDS, a calcium activated chloride channel blocker and BAPTA‐AM, an intracellular calcium channel blocker. The cAMP stimulated Cl− secretion was not altered by administration of this compound demonstrating it does not activate the CFTR mediated Cl− secretion, only the Ca2+ activated Cl− secretion. Using a Xenopus oocyte expression system, where the oocytes express the ANO1 (TMEM16A) CaCC channel endogenously, we confirmed that both the crude and purified fractions of the C. sativus extract activate the ANO1 channel. The molecular size and fragmentation pattern of three compounds were identified by mass spectrometry. To discern the mechanism of the ANO1 activation we measured intracellular Ca2+ levels in human bronchial epithelial cells (IB3‐1) derived from a cystic fibrosis patient, and found the active fraction of the C. sativus extract increases intracellular Ca2+, thereby activating ANO1 channels and Cl− secretion. Together, our data provides evidence that purified extract from C. sativus has potential therapeutic value for treatment of cystic fibrosis and gastrointestinal disorders like constipation by activating ANO1 mediated Cl− secretion in epithelial cells.

Support or Funding Information

Indian Council of Medical Research and Dept. of Biotechnology, Govt. of India
Diaphragm Assessment in Mice Overexpressing Phospholamban in Slow‐Twitch Type I Muscle Fibers
Authors: Chambers Paige J. , Fajardo Val Andrew , Tupling Russell
Published: November 2023

Phospholamban (PLN) is a small transmembrane protein that regulates the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) pump through direct physical interactions. Recent work published by our laboratory revealed that when PLN was transgenetically overexpressed in mice (PlnOE), the postural hind limb muscles presented a phenotype for human Centronuclear Myopathy (CNM). Specifically, PlnOE soleus and gluteus minimus muscles displayed: centrally located oxidative activity, a predominance of hypotrophied type I fibers, and an abundance of central nuclei. To further assess this novel model for CNM, we examined the diaphragm muscles of PlnOE mice, since studies assessing respiratory or diaphragm function in animal models of CNM were limited. Interestingly, our unpublished results suggest that unlike the soleus and gluteus minimus, diaphragm muscles did not display the main characteristics of CNM. Rather than exhibiting type I fiber predominance, the diaphragm muscles exhibited lower type I and greater type II fiber counts, which is of importance since the PlnOE mice were designed to overexpress PLN specifically in their type I fibers. Here, we sought to determine why the PlnOE diaphragm failed to exhibit type I fiber predominance similar to that seen in the soleus and gluteus minimus muscles. Specifically, we focused on calciuneurin (CnA), a Ca2+ ‐dependent phosphatase, and its downstream effector target, nuclear factor of activating T‐cell (NFAT), that upon dephosphorylation by CnA, translocates to the nucleus and acts as a transcription factor for myosin heavy chain I (MHCI). We found that CnA content was significantly higher in both soleus (p=0.02) and gluteus minimus (p=0.05) muscles of PlnOE mice compared to wild type animals. Corresponding with this, nuclear NFAT levels were significantly increased in the gluteus minimus (p=0.01) and trending towards a significant increase in soleus muscle (p=0.07). In contrast, there were no differences between WT and PlnOE in CnA (p=0.23) or nuclear NFAT (p=0.42) content in diaphragm, which suggests the differences in fiber type transition and the CNM phenotype across muscles may be associated with the CnA‐NFAT pathway and calcium signaling. Together these data suggest that the diaphragm of the PlnOE mice may have some undetermined protective mechanism for better regulating cytosolic calcium levels and fiber type expression. Uncovering the mechanisms behind this resistance within the diaphragm of PLNOE mice may aid in exploring new therapeutic strategies for CNM.
Sarcolipin Ablation Impairs Muscle Regeneration After Acute Injury
Authors: Bellissimo Catherine Anne , Bombardier Eric , Chambers Paige , Fajardo Val Andrew , Tupling Russ
Published: November 2023

Sarcolipin (SLN) is a small transmembrane protein that regulates the sarco(endo)plasmic reticulum calcium ATPase (SERCA) pump in muscle. SLN is highly expressed in embryonic muscle and is involved in the regulation of myogenesis. SLN is also upregulated in mouse dystrophic muscles but the role of SLN in muscular dystrophy is unknown. Since muscular dystrophy is characterized by high rates of muscle degeneration/regeneration, one hypothesis is that SLN may be important in the regeneration process but this has not been examined specifically. Here, we used an acute cardiotoxin injury model and Sln‐null mice to determine SLN's role in muscle regeneration. The tibialis anterior of 30 mice, 15 wild type (WT) and 15 Sln‐null mice were injected with cardiotoxin and sacrificed at day 7, 14 and 21 post injury. Muscle homogenates and sections were prepared and used for immunoblotting and immunofluorescence analyses, respectively. Interestingly, ectopic expression of SLN was seen in WT animals only at 21 days post injury. Correspondingly, Sln‐null mice showed elevated centralized nuclei, a marker of regenerating fibers, specifically at day 21 post injury compared to their WT counterparts (2.7 ± 1.0 ×10−4 vs. 3.8 ± 0.6 ×10−4, per regenerating area (μm2), p=0.03). In addition, the average cross‐sectional area of the regenerating myofibers was significantly smaller in the Sln‐null mice only at day 21 post injury compared to WT (1925 ± 168 μm2 vs. 1471 ± 57 μm2, p =0.01 ). Taken together, the failure to clear central nuclei and increase myofiber size at day 21 post injury indicates that SLN ablation delays the later stages of muscle regeneration.

Support or Funding Information

This work was supported by the Canadian Institutes of Health Research (CIHR; MOP 86618 and MOP 47296 to A.R.T).