bluuebunny/crossref_metadata_2025_split huggingface dataset:
SGLT1 and Osmotic Water Flow across the Small Intestine
Authors: Her Ka , Gorraitz Edurne , Loo Donald , Wright Ernest M 
Published: November 2023

Passive water flow is important in the physiology of the small intestine in response to osmotic gradients across the epithelium following the ingestion of food and water; and malabsorption syndromes. However, the mechanism of osmotic water flow across the small intestine is poorly understood. Questions arise on the relative contributions of the cellular and paracellular pathways; and the identity of water channels in the apical and basolateral membrane of the epithelium. It is often assumed that the tight junctions are highly permeable to water simply because they are permeable to small ions; and orthodox aquaporins are not expressed in the small intestine. We have previously postulated that the intestinal Na+/glucose cotransporter SGLT1 plays a key role in fluid transport based on studies of SGLT1 expressed in Xenopus laevis oocytes (see Loo et al 1999. J. Physiology 518:195–202; and Loo et al 1996 PNAS 93:13367–13370). The osmotic water permeability of SGLT1 in oocytes (Pos) is independent of the magnitude and direction of the osmotic gradient, and the presence of ligands (Na+ and sugar), but is inhibited by phlorizin in the presence of Na+ (Ki 5 μM). Furthermore, osmotic water flow has a low temperature dependence (5 kcal/mole) relative to Na+/glucose cotransport (>20 kcal/mole).To investigate the importance of SGLT1 in osmotic flow we measured the effect of phlorizin on osmotic flow. Our approach was to prepare everted sacs of mouse jejunum and measure water flow by recording the change in weight of the sacs with time in response to osmotic gradients. The osmotic gradients were generated by the addition of mannitol to the mucosal buffer, and the contribution of SGLT1 to the osmotic flow was determined by the addition of phlorizin. The experiments were conducted at 21–23°C to maintain the viability of the tissue, a long standing practice in studies of mammalian tissues in vitro. In the absence of an osmotic gradient between the mucosal and serosal solutions there was no significant change in the weight of the sac but the addition of 100 mM mannitol to the mucosal fluid produced a loss of weight equivalent to 16 ± 2 nl min−1 cm−1 and a Pos of 0.012 cm s−1. Phlorizin (250 μM) added to the mucosal fluid reduced the flow to 6.5 ± 2 nl min−1 cm−1.These results indicate that 65% of the osmotic flow across the mouse small intestine occurs through SGLT1, and less than 35% of the total flow occurs through the paracellular pathway.Support or Funding InformationNIH NIDDK DK19567
Ablation of the Renal Urea Transporters UT‐A1 and UT‐A3 Replicates the Benefits of a Low‐protein Diet in Attenuating Diabetic Nephropathy
Authors: Wang Yirong , Himmel Nathaniel J. , Mallow G. Michael , Sun Michael A. , Borchart Matthew R. , Blount Mitsi A. 
Published: November 2023

Diabetic nephropathy (DN) is a leading cause of kidney failure worldwide. Progression of DN can be slowed with a low‐protein diet; however, Western diets exceed the amount of protein physiologically needed, thus excess urea is generated and excreted by the kidney. Urea is reabsorbed in the inner medulla by the urea transporters UT‐A1 and UT‐A3. Although regulation of urea handling by these transporters controls urine concentration, the impact of these transporters on DN has not been explored. We induced DN in wild type (WT) and UT‐A1/A3 null mice (KO) using low‐dose streptozotocin injections and fed the mice a low (LP; 14%)‐, normal (NP; 25%)‐ or high (HP; 40%)‐protein diet over 10 wk. All DN mice demonstrated glycosuria and polyuria regardless of diet or genotype. Creatinine clearance was dampened in DN WT mice on LP and NP diets and was significantly decreased in DN WT mice on a HP diet. Interestingly, DN KO mice did not have diminished creatinine clearance rates regardless of diet suggesting the inability to reabsorb urea preserves renal function in DN. Histological analysis showed advanced interstitial fibrosis in WT DN mice fed a NP and HP diet; however, diabetic KO mice on the same diets had minimal fibrosis. Both DN and high urea levels are associated oxidative stress, prompting us to investigate multiple markers of oxidative stress. We found that DN KO mice fed HP diet were protected from the oxidative stress observed in DN WT mice on the same diet. Our data indicate in the absence of UT‐A1 and UT‐A3, the collecting duct fails to reabsorb the excess urea from a high protein diet and in doing so, prevents the interstitial fibrosis that occurs as a consequence of increased oxidative stress in the diabetic kidney.Support or Funding InformationHHMI Science Education Program Award #52006923
Tight junction protein abundance is altered in Metformin treated airway epithelial cells
Authors: Kalsi Kameljit K , Garnett James P , Baker Emma H , Baines Deborah L 
Published: November 2023

Airway epithelial tight junction (TJ) proteins form a resistive barrier to the external environment, however during respiratory bacterial infection TJ abundance is altered. Metformin used for the treatment of diabetes is known to increase transepithelial resistance (TEER) of airway epithelial cells. We investigated the effect of metformin and Staphylococcus aureus inoculation on TJ abundance and the assembly/reassembly of the TJ protein in airway epithelial cells. Occludin protein was detected by immunoblot analysis with a major protein band at 60 kD with cleavage products at 46, 44 and 37 kD in human airway epithelial cells (H441), pre‐treatment with 1 mM metformin for 18h, resulted in an increase in the 60 kD protein band (13 ± 7%, P<0.05; n=4) and a decrease in the cleavage product at 44 kD (37 ± 4%, P<0.05; n=4). Inoculating H441 monolayers with S.aureus produced a significant reduction in abundance in both 60 and 44 kD protein bands (35 ± 11% and 41 ± 5% respectively, P<0.05; n=4) which was not reversed with metformin pre‐treatment. Similarly occludin and Z0‐1 abundance were both enhanced with metformin and reduced with S.aureus in immunostained epithelial monolayers. Disassembly and reassembly of TJs in the presence of a calcium chelating agent was not protected or enhanced by metformin pre‐treatment. These data provide evidence that metformin regulates TJ protein abundance through a mechanism that does not involve changes in assembly.Support or Funding InformationMRC‐MICA MR/K012770/1
Dehydration Impairs Synaptic Plasticity by Interfering with Late‐Phase Long‐Term Potentiation (L‐LTP)
Authors: Vashisht Anirudh , Morykwas Michael , Hegde Ashok , Argenta Louis , McGee Maria 
Published: November 2023

Dehydration impairs cognitive function. To explore the possible mechanisms, we dehydrated mouse brain tissue and measured LTP, the best understood cellular process of synaptic plasticity that underlies learning and memory in vertebrates.We determined physiologic hydration by titrating water activity in mouse brain explants. Specifically, we immersed the 60–90‐mg explants in baths of artificial cerebrospinal fluid (ACSF) equilibrated at room temperature in which inert, non‐penetrating polymer polyethylene glycol 8000 was dissolved to attain colloidosmotic pressures of 0 mmHg (in control ACSF), 54 mmHg, 101 mmHg, and 196 mmHg. We then calculated the parameters reflecting brain hydration, including hydration potential and hydraulic conductance, from initial flow rates determined gravimetrically. To explore the effect of dehydration on synaptic plasticity, hippocampus slices were equilibrated for 30 min at the above pressures prior to recording field excitatory postsynaptic potentials (fEPSP). Parameters for basal synaptic transmission were derived from input‐output tracings following incremental stimuli. In a separate set of slices, L‐LTP was induced by spacing four 100‐Hz trains 5‐min apart and recording the results for 3 h. Water activity titrations were conducted at room temperature and LTP at 32 °C. All experiments were reproduced in at least 6 mice; results are presented as means and standard errors.Fluid transferred either to or from the brain explants, depending on the bath pressure. Initial fluid transfer rates—3.486 ± 0.1919, 0.08950 ± 0.3907, −1.820 ± 1.258 and −4.248 ± 2.338 μl/min/g— increased linearly ( r2> 0.9, by linear regression analyses) with nominal bath pressures of 0, 54, 101, and 196 mmHg, respectively. The hydraulic conductance, calculated from the slope of the fitted regression lines, was 0.051 ± 0.0019 μl/min/g/mmHg, and the hydration potential, calculated from the pressure at initial rate = 0, was 56.44 ± 4.96 mmHg. In slices equilibrated at near this physiologic hydration potential, basal synaptic transmission resembled that of the slices in control ACSF. However, dehydrating the slices by increasing the colloid osmotic pressure increased basal synaptic transmission, indicating neuronal hyper‐excitability. Similarly, L‐LTP induced at the 54 mmHg level did not differ from that in controls, but progressive dehydration impaired its induction. Induction was completely abolished at a nominal pressure of 196 mmHg. The surprisingly high hydration potential measured in the explanted brain tissue indicates lower water activity in the brain interstitial matrix than in cerebrospinal fluid (CSF) and reflects competition for water in the crowded brain microenvironment. As a notable consequence, neuronal excitability is highly sensitive to changes in water activity. Our findings predict that local dehydration affects cognitive function and impairs synaptic plasticity by preventing L‐LTP induction.
The Anti‐Inflammatory Peptide Ac‐SDKP is Released from Thymosin β4 by Meprin α and Prolyl Oligopeptidase
Authors: KUMAR NITIN , Nakagawa Pablo , Janic Branislava , Romero Cesar A. , Worou Morel E. , Peterson Edward L. , Ongeri Elimelda E. , Niyitegeka Jean‐Marie V. , Rhaleb Nour‐Eddine , Carretero Oscar A. 
Published: November 2023

N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (Ac‐SDKP) is a natural tetrapeptide with anti‐inflammatory and anti‐fibrotic properties. We have previously shown that prolyl oligopeptidase (POP) is involved in the Ac‐SDKP release from thymosin β4 (Tβ4). However, POP can only hydrolyze peptides shorter than 30 amino acids and Tβ4 is 43 amino acids long. This suggests that Tβ4 must be hydrolyzed by another peptidase that releases N‐terminal intermediate peptide(s) <30 amino acids before POP hydrolysis takes place. Our search in peptidase database for potential candidate(s) gave high score for meprin α metalloprotease. Therefore, we hypothesize that Tβ4 is hydrolyzed by meprin α prior to POP hydrolysis. To test this, in vitro and in vivo studies were performed. In vitro, we found that the incubation of Tβ4 with both, meprin α and POP releases Ac‐SDKP, whereas it failed when Tβ4 is incubated with either meprin α or POP alone. Incubation of Tβ4 with rat kidney homogenate increases Ac‐SDKP, which is blocked by actinonin (meprin α inhibitor). In addition, kidney from meprin α knockout mice showed a significantly lower Ac‐SDKP as compared to its wild type control. In vivo, we observed that rat treated with captopril (ACE inhibitor) increased plasma concentrations of Ac‐SDKP, which is inhibited by the co‐administration of actinonin (vehicle 3.1±0.22 nmol/L; captopril 15.1±0.7 nmol/L; captopril + actinonin 6.1±0.3 nmol/L; vehicle versus captopril, P<0.002; captopril versus captopril + actinonin, P<0.002). Similar results were obtained with urinary Ac‐SDKP excretion. We conclude that Ac‐SDKP is released from Tβ4 by the successive action of meprin α and POP.Support or Funding InformationThis work is supported by National Institute Health Grant HL028982
Involvement of the Cyclic AMP Pathway in Dendritic Cell Regulation of Th2 Immune Responses
Authors: Chinn Amy M , Lee Jihyung , Herdman Scott , Raz Eyal , Insel Paul A 
Published: November 2023

Dendritic cells, the main antigen‐presenting cells, are involved in the induction of type II helper T cell (Th2) immune responses, but the mechanism by which this occurs is not fully elucidated. Mice that have a CD11c‐specific deletion of the Gnas gene (CD11cΔGnas), which encodes the Gαs protein of the heterotrimeric (αβγ) Gs signaling protein complex, display an allergic asthma phenotype; the corresponding decrease in cyclic AMP (cAMP) in dendritic cells biases them to provoke a Th2 response ex vivo and in vivo (Lee et al PNAS 112: 1529–34 [2015]). The molecular events involved in the Th2 bias that results from the increased Gαi:Gαs signaling ratio in this setting remain to be defined. Here, we compare the expression of G protein‐coupled receptors (GPCRs), including those that regulate cAMP formation, and the expression of other components that regulate cAMP levels of wild‐type (WT) and CD11cΔGnas mouse bone marrow‐derived dendritic cells (BMDCs). Quantitative PCR (qPCR)‐based Taqman GPCR arrays revealed that WT BMDCs express 120 non‐chemosensory GPCRs while CD11cΔGnas BMDCs express 145 GPCRs. Notably, GPCRs that couple to Gαi (rather than other Gα proteins) have the greatest increase in gene expression in the CD11cΔGnas BMDCs compared to BMDCs from WT mice. Moreover, CD11cΔGnas BMDCs have reduced expression (as assayed by qPCR; p<0.05) of cyclic nucleotide phosphodiesterase (PDE) isoforms that degrade cAMP and are known to be induced by increases in cellular cAMP levels (PDE's1B, 3B, 4A, 4B, 4D, and 7B). PDE4B is also the predominant PDE isoform in mouse BMDCs. In addition, we found a significant (p<0.05) change in the mRNA expression of certain adenylyl cyclase isoforms: an increase in ADCY3 and decrease in ADCY4. Together, these data indicate that the expression of GPCRs (in particular Gαi‐coupled GPCRs) and other components that regulate cAMP levels are altered in dendritic cells that promote Th2 bias. Such GPCRs and post‐G protein components may contribute to the greater Th2 responses and allergic asthma observed in CD11cΔGnas mice.Support or Funding InformationSupported by NIH grants R56AI110505 and T32GM007752.
Role of calcium signaling for GDNF secretion, ureter branching and early nephron formation
Authors: Fontana Jacopo Maria , Jess David Unnersjö , Blom Hans , Brismar Hjalmar , Aperia Anita 
Published: November 2023

Low nephron endowment has an adverse effect on the outcome of many kidney diseases and is a risk factor for chronic kidney disease. The formation of nephrons occurs as a result of the reciprocal interaction between metanephric mesenchymal (MM) cells and the ureteric bud (UB). MM cells secrete the trophic factor GDNF, which binds to the Ret receptors on the UB. Signaling via GDNF and the Ret receptor promotes UB branching and plays a central role for initiating nephron formation. The mechanisms controlling the release of GDNF from the MM cells has been elusive, despite its fundamental importance for kidney development. Most secretory processes are controlled by calcium signaling. Here we demonstrate, using embryonic E14 rat kidneys cultured for 1–2 days, the occurrence of spontaneous calcium transients in MM cells with a frequency of 2–29 min−1. Depletion of calcium stores in the endoplasmic reticulum (ER) with cyclopiazonic acid (CPA) caused drastic reduction of calcium activity, while acute exposure of kidneys to calcium‐free extracellular solution had no effect. To chronically reduce the ER calcium stores and downregulate the spontaneous calcium activity in MM cells, kidneys were exposed to low concentration of CPA from DIV1 to DIV2. This resulted in swelling and significant reduction of UB as well as of significant reduction of UB branching points and of newly formed pre‐glomeruli. In addition, GDNF concentration in the culture media was significantly lower for CPA treated than for control kidneys. To further assess the role of calcium signaling for GDNF secretion, we stained the kidneys for MM and UB markers (WT‐1 and E‐Cadherin respectively) and for GDNF, as well as synaptotagmin‐1 (SYT1), a calcium binding protein that is the major calcium sensor activating secretory processes in the brain. By applying a tissue clearing protocol and super‐resolution microscopy, we could resolve the localization of SYT1 and GDNF in the MM cells, both surrounding the UB tips. In summary we have identified a novel calcium dependent link in the complex process of nephron formation. In the light of this, our results indicate that the vesicle transport and calcium‐regulated fusion that control neurotransmitter release at the synapsis could also have fundamental importance for early embryonic development of methanephric kidney.Support or Funding InformationThe Swedish Research Council (AA and HB).
A Mathematical Model of Calcium and cAMP Signaling in Pulmonary Microvascular Endothelial Cells
Authors: Webb Kristal J , Wiles C Alex , Annamdevula Naga , Sweat Rachel , Britain Andrea L , Phan Anh‐Vu , Townsley Mary I , Leavesley Silas J , Rich Thomas C 
Published: November 2023

The goal of this project is to understand how the spatial distribution of intracellular Ca2+ alters the kinetics and spatial spread of agonist‐induced cAMP signals in pulmonary microvascular endothelial cells (PMVECs). PMVECs predominantly contain the Ca2+‐inhibited adenylyl cyclase type 6 (AC6) and also contain low levels of the Ca2+‐stimulated phosphodiesterase type 1 (PDE1). It has been clearly demonstrated that Ca2+ influx through store operated Ca2+ (SOC) channels inhibits AC6 activity in PMVECs. Our preliminary data indicate that Ca2+ influx through T‐type Ca2+ channels and TRPV4 cation channels inhibits agonist‐induced cAMP production. We have developed mathematical models to investigate how the spatial reach of Ca2+ influx through each of these channels alters agonist‐induced cAMP signals. The models describing this intracellular feedback network were developed in the MATLAB programming environment. Our initial models used ordinary differential equations to describe the kinetics and spatial redistribution of Ca2+ and cAMP signals between intracellular compartments. Based upon experimental observations, Ca2+ channels were distributed in three discrete subplasmalemmal compartments. In addition, a fourth cytosolic compartment was incorporated into the model. This model also describes Ca2+ and cAMP buffering as well as Ca2+ efflux. A Runge‐Kutta solver was used to perform numerical integration. Simulations of the model predict that at low agonist levels, low levels of Ca2+ influx, Ca2+ signals do not readily spread between compartments. Similarly, the model predicts that agonist‐induced cAMP signals do not readily spread between compartments. Thus, Ca2+ influx in one compartment leads to localized inhibition of AC6 and a localized decrease in cAMP. We conclude that at low agonist concentrations, activation of different populations of Ca2+ channels regulates different pools of AC6 thus cAMP levels in different subcellular compartments. However, at higher and/or prolonged agonist concentrations, Ca2+ signals spread between intracellular compartments, increasing the range of AC6 inhibition. We are currently extending this model to more accurately describe the relative diffusion of second messengers using a finite element analysis approach.Support or Funding InformationThis work was supported by NIH P01HL066299 and T32HL076125.
A‐Kinase Anchoring Proteins Coordinate the Contractile Phenotype of Airway Smooth Muscle
Authors: Poppinga Wilfred Jelco , Han Bing , Elzinga Carolina R. , Halayko Andrew J. , Meurs Herman , Schmidt Martina 
Published: November 2023

Smooth muscle contraction is modulated by three pathways: the amount of free [Ca2+]i; Ca2+ sensitivity; and, the regulation of intracellular filaments comprising the contractile machinery. Recently, a new dimension has emerged to play a role in airway smooth muscle function, namely the scaffolding protein family A‐kinase anchoring proteins (AKAPs) that bind cAMP effector PKA and other proteins to specific cellular compartments. Here we studied the role of AKAPs in maintaining the contractile phenotype of the airway smooth muscle. Treatment with AKAP‐PKA interaction inhibitor st‐Ht31 (50 μM) for 96 h caused an increase in abundance of the contractile proteins α‐smooth muscle actin (αSMA) (283% of basal) and calponin (159% of basal) in immortalized human tracheal smooth muscle cells, without an increase in mRNA expression or stability. To understand potential underlying mechanisms for this response, after 72 h incubation with st‐Ht31, we treated cells (24 h) with the mRNA transcription inhibitor, actinomycin D (1 μg/mL): this decreased abundance of both proteins in absence and presence of st‐Ht31 (~50 αSMA, ~70% calponin). Nonetheless, st‐Ht31 still induced higher protein levels compared to untreated control cultures (~35% αSMA, ~80% calponin). Inhibition of protein synthesis using cycloheximide (5 μg/mL) had no effect on the expression αSMA, however it did increase the expression of calponin under both control and st‐Ht31‐treatment conditions (~200%), suggesting that αSMA is a rather stable protein and that calponin degradation requires de novo synthesis of an unknown protein. Lysosomal inhibition using chloroquine (50 μM) did not affect either protein. Inhibition of the proteasome with MG‐132 (5 μM) reversed st‐Ht31‐induced increase of αSMA (102% of basal), without affecting the st‐Ht31 induced increase of calponin (210% of basal), revealing that st‐Ht31‐induced increase in αSMA requires proteasomal degradation of an unknown protein. Functionally, incubation of human tracheal smooth muscle strips with st‐Ht31 for 96 h increased both the abundance of contractile proteins and the contractile response to methacholine. These data implicate that AKAPs regulate the maintenance of contractile smooth muscle, probably by post‐translationally controlling levels of contractile proteins.Support or Funding InformationThis study was supported by the Dutch Lung Foundation (Grant #: 3.2.11.015) and an Ubbo Emmius grant.