Douglas Charles Eaton

Introduction: Although both COVID-19 and non-COVID-19 ARDS can be accompanied by significantly increased levels of circulating cytokines, the former significantly differs from the latter by its higher vasculopathy, characterized by increased oxidative stress and coagulopathy in lung capillaries. This points towards the existence of SARS-CoV2-specific factors and mechanisms that can sensitize the endothelium towards becoming dysfunctional. Although the virus is rarely detected within endothelial cells or in the circulation, the S1 subunit of its spike protein, which contains the receptor binding domain (RBD) for human ACE2 (hACE2), can be detected in plasma from COVID-19 patients and its levels correlate with disease severity. It remains obscure how the SARS-CoV2 RBD exerts its deleterious actions in lung endothelium and whether there are mechanisms to mitigate this.Methods: In this study, we use a combination of in vitro studies in RBD-treated human lung microvascular endothelial cells (HL-MVEC), including electrophysiology, barrier function, oxidative stress and human ACE2 (hACE2) surface protein expression measurements with in vivo studies in transgenic mice globally expressing human ACE2 and injected with RBD.Results: We show that SARS-CoV2 RBD impairs endothelial ENaC activity, reduces surface hACE2 expression and increases reactive oxygen species (ROS) and tissue factor (TF) generation in monolayers of HL-MVEC, as such promoting barrier dysfunction and coagulopathy. The TNF-derived TIP peptide (a.k.a. solnatide, AP301) -which directly activates ENaC upon binding to its a subunit- can override RBD-induced impairment of ENaC function and hACE2 expression, mitigates ROS and TF generation and restores barrier function in HL-MVEC monolayers. In correlation with the increased mortality observed in COVID-19 patients co-infected with S. pneumoniae, compared to subjects solely infected with SARS-CoV2, we observe that prior intraperitoneal RBD treatment in transgenic mice globally expressing hACE2 significantly increases fibrin deposition and capillary leak upon intratracheal instillation of S. pneumoniae and that this is mitigated by TIP peptide treatment.

We examined the interaction of a membrane-associated protein, MARCKS-like Protein-1 (MLP-1), and an ion channel, Epithelial Sodium Channel (ENaC), with the anionic lipid, phosphatidylinositol 4, 5-bisphosphate (PIP₂). We found that PIP₂ strongly activates ENaC in excised, inside-out patches with a half-activating concentration of 21 ± 1.17 µM. We have identified 2 PIP₂ binding sites in the N-terminus of ENaC β and γ with a high concentration of basic residues. Normal channel activity requires MLP-1’s strongly positively charged effector domain to electrostatically sequester most of the membrane PIP₂ and increase the local concentration of PIP₂. Our previous data showed that ENaC covalently binds MLP-1 so PIP₂ bound to MLP-1 would be near PIP₂ binding sites on the cytosolic N terminal regions of ENaC. We have modified the charge structure of the PIP₂ –binding domains of MLP-1 and ENaC and showed that the changes affect membrane localization and ENaC activity in a way consistent with electrostatic theory.

We examined the interaction of a membrane‐associated protein, MARCKS‐like Protein‐1 (MLP‐1), and an ion channel, Epithelial Sodium Channel (ENaC), with phosphatidylinositol 4, 5‐bisphosphate (PIP2). PIP2 activates ENaC in inside‐out patches (half activating concentration of 21 ± 1.17). There are several PIP2 binding sites on ENaC, but 2 binding sites with a large number of basic residues at the N‐terminus of ENaC b and g are critical sites for PIP2 regulation of ENaC. PIP2 is necessary to open ENaC, but there is a problem with a simple model of ENaC and PIP2 associating by membrane lateral diffusion. ENaC is a relatively rare protein (a few functional apical channels/m2). PIP2 is also a rare molecule: less than 1 in 1000 membrane lipids. Given the known abundance of PIP2 and ENaC and the diffusion constant of PIP2 in the apical membrane, the mean time it would take a PIP2 molecule to find an ENaC channel by random lateral diffusion is 6.3x102s or approximately 10 minutes. But, in normal epithelial cells expressing ENaC, the channel opens about every 2 seconds. We hypothesize that normal channel activity requires MLP‐1 associated with the inner leaflet of the cell membrane. MLP‐1’s strongly positively charged effector domain sequesters PIP2 electrostatically without direct covalent binding. This implies differences between PIP2 association with MLP‐1 and the binding of a ligand to a specific receptor. First, the effect of MLP‐1 charge acts at a distance. The coulombic force exerted by MLP‐1 on PIP2 does decrease as the square of the distance between MLP‐1 effector domain and PIP2, but the distances within the membrane are relatively small and the membrane dielectric constant is also very small so the forces can be significant over a large membrane area. In the absence of MLP‐1, PIP2 moves around the membrane lipid in a two‐dimensional random walk, but the presence of the charged effector domain biases the walk so that PIP2 always moves towards MLP‐1, at first slowly and then rapidly until it is in MLP‐1’s potential well. This implies that MLP‐1 increases the local concentration of PIP2 exponentially as electrostatic theory predicts. MLP‐1 membrane concentration is near 10mM which is comparable to the concentration of PIP2. Given PIP2‘s lateral mobility in the membrane of about 10‐8cm2s‐1, MLP‐1 would rapidly sequester most membrane PIP2. This would increase the local PIP2 concentration over a hundred‐fold. Our data shows that ENaC covalently binds MLP‐1, so this increased PIP2 concentration would be in close proximity to PIP2 binding sites on the cytosolic N terminus of ENaC. This binding is also electrostatic since the ENaC binding sites contain multiple basic residues. The advantage of electrostatic interaction is that PIP2 can remain in close proximity to both ENaC and MLP‐1. We have modified the charge structure of the PIP2 –binding domains of MLP‐1 and ENaC and showed that the changes affect membrane localization and ENaC activity consistent with electrostatic theory. Using STED FRAP, we also measured the diffusion rates of MLP‐1, ENaC, and PIP2 and ENaC and PIP2 in the absence of MLP‐1 that also support an electrostatic model.

Normal gas exchange in the alveoli is facilitated by a thin fluid layer. This layer is carefully regulated by two ion channels: highly selective channels (HSC or ENaC) and nonselective cation channels (NSC). Previous research found that both channels contribute about equally to alveolar fluid clearance (AFC). Since the molecular composition of NSC is a recent discovery, not much is known about NSC’s response to various respiratory pathogens. NSC are composed of an alpha‐ENaC subunit and at least one acid‐sensing ion channel 1a (ASIC1a) subunit. Our study aims are to understand how NSC contribute to alveolar epithelial permeability and how the lungs respond to toxins from respiratory pathogens. We hypothesize that AFC will be lower in the absence of ASIC1a (ASIC1 KO) when exposed to toxins from respiratory pathogens: Lipopolysaccharide (LPS) from E. coli and Pneumolysin (PLY) from P. pneumoniae. Additionally, we hypothesize that ASIC1 KO animals will have increased baseline alveolar epithelium permeability. All animal experiments were approved by Emory’s IACUC. We introduced pathogen toxins via intratracheal instillation to ASIC1 KO (KO) and wildtype (WT) mouse models (both on a C57Bl6 background). After a four‐hour incubation period, we euthanized the mice by isoflurane overdose. We determined AFC by instilling their lungs with 0.7ml of a 2.0% Evans Blue (EB) or Fluorescein isothiocyanate–dextran (FITC) solution via the trachea. We took samples at t=0 and t=30 minutes after instillation. We determined the EB/ FITC concentration of both samples using spectrophotometry and calculated AFC from the change in dye concentration. Our preliminary findings suggest that LPS and PLY inhibit all AFC associated with HSC leaving only AFC associated with NSC. The AFC of LPS exposed KO animals is low compared to AFC for LPS exposed WT animals (6.04 ± 3.02, n= 15 vs 15.8 ± 5.61, n= 11, p< 0.001). Similarly, AFC of PLY exposed KO mice is lower than AFC for PLY exposed WT mice (2.78 ± 0.96, n= 10 vs 12.4 ± 4.36, n= 4, p< 0.001). In the toxin exposed KO groups, neither HSC nor NSC are fully functioning or contributing to AFC. We are still collecting data on the permeability of the alveolar epithelium. There is evidence to suggest that the regulation of HSC and NSC are drastically different. Given the critical nature of the alveoli fluid layer, it would be logical that NSC channels serve as a compensatory mechanism when HSC are inhibited by pathogens. These findings can help guide treatment for alveolar flooding caused by toxins from different respiratory pathogens.

Hypertension (HTN) is an inflammatory disease which can be attributed to excessive sodium (Na ⁺ ) reabsorption by cells in the aldosterone‐sensitive distal nephron (ASDN), resulting in decreased water excretion and a systemic increase in blood pressure. The need for rapid identification of new therapies is important, since anti‐hypertensive drugs are often unsuccessful in regulating blood pressure. Interleukin‐6 (IL6) levels are consistently increased in plasma from hypertensive individuals. Additionally, our previous data suggests that IL6 can activate the mineralocorticoid receptor (MR) both in vitro and in vivo , as well as increase blood pressure. However, the exact intracellular signaling pathways are unknown. We first utilized RNA sequencing (RNAseq) to uncover novel targets. These studies used the mDCT15 cell model for the DCT2; cells were treated with IL6 (100ng/mL, 24 hours) or vehicle (n=4, each), then total RNA isolated. Data from the Illumina‐sequenced transcriptome underwent comparative analysis to identify significant differentially expressed transcripts between vehicle and IL6 treated cells. These experiments revealed an intricate pathway for the family of mitogen‐activated protein kinases (MAPKs). We observed significant reductions in Map3k11 (padj, 0.01), along with Mapk8ip3 (padj, 0.02) expression following IL6 treatment. Both are important modulators of the JNK pathway. Map3k11 , also known as JNK‐activator, MLK3. Mapk8ip3 encodes for JNK‐scaffolding activator, JIP3. We hypothesized that the MAPK pathways are a downstream regulator of IL6. Using total protein from IL6 treated cells (15‐30min), we observed a strong increase in pERK1/2 expression, with no changes in total ERK1/2 levels. To explore the role for ERK1/2 in ENaC activation, we measured transepithelial voltage and resistance using a voltohmmeter, and calculated current in mDCT15 cells (n=3/group) following IL6 treatment (1hr) and with an ERK1/2 inhibitor. IL6 increased amiloride‐sensitive current, which was reduced with ERK1/2 inhibition. Together, these data suggest an immediate increase in pERK1/2 activation, which was followed with an increase in ENaC activation. Interestingly, we observed an inhibition in the JNK‐pathway members with 24hr IL6 stimulation, suggestion a negative feedback regulation. These data support a time‐sensitive role for IL6‐mediated ENaC activation via MAPKs in the distal tubule and represent novel targets for alternate IL6‐mediated MR activation.

The epithelial sodium channel (ENaC) regulates blood pressure by fine-tuning distal nephron sodium reabsorption. Our previous work has shown that ENaC gating is regulated by anionic phospholipid phosphates, including phosphatidylinositol 4,5-bisphosphate (PIP₂). The PIP₂-dependent regulation of ENaC is mediated by the myristoylated alanine-rich protein kinase C substrate-like protein-1 (MLP-1). MLP-1 binds to and is a reversible source of PIP₂ at the plasma membrane. We examined MLP-1 regulation of ENaC in distal convoluted tubule clonal cell line DCT-15 cells. Wild-type MLP-1 runs at an apparent molecular mass of 52 kDa despite having a predicted molecular mass of 21 kDa. Native MLP-1 consists of several distinct structural elements: an effector domain that is highly positively charged, sequesters PIP₂, contains serines that are the target of PKC, and controls MLP-1 association with the membrane; a myristoylation domain that promotes association with the membrane; and a multiple homology 2 domain of previously unknown function. To further examine MLP-1 in DCT-15 cells, we constructed several MLP-1 mutants: WT, a full-length wild-type protein; S3A, three substitutions in the effector domain to prevent phosphorylation; S3D mimicked constitutive phosphorylation by replacing three serines with aspartates; and GA replaced the myristoylation site glycine with alanine, so GA could not be myristoylated. Each mutant was tagged with either NH₂-terminal 3XFLAG or COOH-terminal mCherry or V5. Transfection with MLP mutants modified ENaC activity in DCT-15 cells: activity was highest in S3A and lowest in S3D, and the activity after transfection with either construct was significantly different from WT. In Western blots, when transfected with 3XFLAG-tagged MLP-1 mutants, the expression of the full length of MLP-1 at 52 kDa increased in mutant S3A-MLP-1-transfected DCT-15 cells and decreased in S3D-MLP-1-transfected DCT-15 cells. Several lower molecular mass bands were also detected that correspond to potential presumptive calpain cleavage products. Confocal imaging shows that the different mutants localize in different subcellular compartments consistent with their preferred location in the membrane or in the cytosol. Activation of protein kinase C increases phosphorylation of endogenous MLP-1 and reduces ENaC activity. Our results suggest a complicated role for proteolytic processing in MLP-1 regulation of ENaC.

Hypertension (HTN) is a major clinical concern. HTN can be attributed to excessive sodium (Na ⁺ ) reabsorption by cells in the aldosterone‐sensitive distal nephron (ASDN), resulting in decreased water excretion and a systemic increase in blood pressure. The need for rapid identification of new therapies is important, since anti‐hypertensive drugs are often unsuccessful in regulating blood pressure. Prior research demonstrates an association between inflammation and the progression of HTN. It has been estimated that high intake of Na ⁺ in the ‘Western’ diet along with increased extracellular Na ⁺ may drive a systemic inflammatory response. These responses include increased cytokines, such as interleukin‐6 (IL6). IL6 has been shown to be consistently increased in hypertensive patients. Additionally, our previous data suggests that IL6 can activate the mineralocorticoid receptor (MR) both in vitro and in vivo , as well as increase blood pressure. Our data also suggest a strong mechanism for reactive oxygen species (ROS)‐ and Rac1‐driven activation of the MR, leading to distal Na ⁺ transporter activity. In this study, we utilized RNA sequencing (RNAseq) revealing novel targets driving the IL6‐mediated activation of the MR in the DCT2. We hypothesize that IL6 induces a specific DCT2 transcriptome. To test this hypothesis, we used mDCT15 cells that model the DCT2; an area critical for MR‐mediated Na ⁺ reabsorption. mDCT15 cells were treated with IL6 (100ng/mL, 24 hours) or vehicle (n=4, each), then total RNA isolated. RNA was then converted to mRNA and used to build a library. Data from the Illumina‐sequenced transcriptome underwent comparative analysis to identify significant differentially expressed transcripts between vehicle and IL6 treated cells. Out of 10849 shared transcripts, 64 were specifically expressed with IL6 treatment (Fig. ). Following Gene Ontology (GO) Enrichment, the gene ratio for ribosomal machinery and cytoskeleton remodeling was significant. We also uncovered several targets of interest which were differentially expressed, as shown by adjusted p value (p‐adj), following IL6 treatment. The genes Gstp1 (5.48×10 ⁻¹⁰ , p‐adj ) and HSP90 (3.03×10 ⁻⁶ , p‐adj ) were both significantly increased following IL6 treatment. Gstp1 encodes for glutathione S‐transferase, which is important in reducing cellular ROS, while Hsp90 is critical for the movement of the MR into the nucleus. Additionally, Arhgef16 (0.0033, p‐adj ) was significantly reduced following IL6 treatment; this protein is a Rho‐guanine exchange factor (RhoGEF) and may be important in the regulation of Rac1. These targets are in line with data from our laboratory suggesting that the IL6‐induced increases in ROS generation and Rac1 activation induce MR translocation and activation. Together, these genes represent novel targets for alternate IL6‐mediated MR activation. Support or Funding Information: DK115660, ASN Gottschalk Award to BMW; DK110409 to DCE Interleukin 6 Stimulated Differential Gene Expression in mDCT15 Cells. Figure 1

Significance Statement: The mineralocorticoid receptor within intercalated cells increases chloride transport in the mouse cortical collecting duct (CCD) through a mechanism involving the apical chloride/bicarbonate exchanger pendrin. In mouse studies, the authors demonstrated that ablating this receptor in intercalated cells markedly changes pendrin’s total abundance and subcellular distribution through a direct effect of the receptor, which occurs over a wide range in serum potassium concentration. The mineralocorticoid receptor within intercalated cells also indirectly modulates sodium channel activity in principal cells. Aldosterone increases pendrin through mechanisms both dependent and independent of the receptor. These data suggest that mineralocorticoid receptor antagonists increase NaCl excretion by the kidney, in part, by inhibiting intercalated cell pendrin-mediated chloride absorption directly and by inhibiting epithelial sodium channel–mediated sodium absorption indirectly through an effect of intercalated cell receptor blockade. Background: Aldosterone activates the intercalated cell mineralocorticoid receptor, which is enhanced with hypokalemia. Whether this receptor directly regulates the intercalated cell chloride/bicarbonate exchanger pendrin is unclear, as are potassium’s role in this response and the receptor’s effect on intercalated and principal cell function in the cortical collecting duct (CCD). Methods: We measured CCD chloride absorption, transepithelial voltage, epithelial sodium channel activity, and pendrin abundance and subcellular distribution in wild-type and intercalated cell–specific mineralocorticoid receptor knockout mice. To determine if the receptor directly regulates pendrin, as well as the effect of serum aldosterone and potassium on this response, we measured pendrin label intensity and subcellular distribution in wild-type mice, knockout mice, and receptor-positive and receptor-negative intercalated cells from the same knockout mice. Results: Ablation of the intercalated cell mineralocorticoid receptor in CCDs from aldosterone-treated mice reduced chloride absorption and epithelial sodium channel activity, despite principal cell mineralocorticoid receptor expression in the knockout mice. With high circulating aldosterone, intercalated cell mineralocorticoid receptor gene ablation directly reduced pendrin’s relative abundance in the apical membrane region and pendrin abundance per cell whether serum potassium was high or low. Intercalated cell mineralocorticoid receptor ablation blunted, but did not eliminate, aldosterone’s effect on pendrin total and apical abundance and subcellular distribution. Conclusions: With high circulating aldosterone, intercalated cell mineralocorticoid receptor ablation reduces chloride absorption in the CCD and indirectly reduces principal cell epithelial sodium channel abundance and function. This receptor directly regulates pendrin’s total abundance and its relative abundance in the apical membrane region over a wide range in serum potassium concentration. Aldosterone regulates pendrin through mechanisms both dependent and independent of the IC MR receptor.

Pulmonary permeability edema is characterized by reduced alveolar Na⁺ uptake capacity and capillary barrier dysfunction and is a potentially lethal complication of listeriosis. Apical Na⁺ uptake is mainly mediated by the epithelial sodium channel (ENaC) and initiates alveolar liquid clearance. Here we examine how listeriolysin O (LLO), the pore-forming toxin of Listeria monocytogenes, impairs the expression and activity of ENaC. To that purpose, we studied how sub-lytic concentrations of LLO affect negative and positive regulators of ENaC expression in the H441 airway epithelial cell line. LLO reduced expression of the crucial ENaC-α subunit in H441 cells within 2 h and this was preceded by activation of PKC-α, a negative regulator of the channel’s expression. At later time points, LLO caused a significant reduction in the phosphorylation of Sgk-1 at residue T256 and of Akt-1 at residue S473, both of which are required for full activation of ENaC. The TNF-derived TIP peptide prevented LLO-mediated PKC-α activation and restored phospho-Sgk-1-T256. The TIP peptide also counteracted the observed LLO-induced decrease in amiloride-sensitive Na⁺ current and ENaC-α expression in H441 cells. Intratracheally instilled LLO caused profound pulmonary edema formation in mice, an effect that was prevented by the TIP peptide; thus indicating the therapeutic potential of the peptide for the treatment of pore-forming toxin-associated permeability edema.