bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationDFG
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 InformationSupported 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 InformationNatural 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 InformationNIH
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 InformationThis 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 InformationNIDDK085097‐RSH2T32DK7656‐21 Emory Renal Division‐BMW
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

IntroductionMitochondrial 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.HypothesisP‐Y of MCU activates mitochondrial Ca2+ uptake and induces mitochondrial Ca2+ overload and cardiomyocyte death.MethodsNon‐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.Resultsa1‐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.ConclusionMCU 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 InformationThis 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 InformationNIH 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 InformationIntramural 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 InformationDr. Enrique Soto EguibarInstitute of Physiology ‐ BUAP A.P. 406Puebla, Pue. Postal code: 72570 MexicoTel: (52‐222) 2295500 est 7316Fax: (52‐222) 2295500 ext 7301