Nonselective Cation Channels Maintain Alveolar Fluid Clearance After Exposure to Toxins from Respiratory Pathogens Open Access

Abstract

Normal gas exchange in the alveoli is facilitated by a thin fluid layer. This layer is carefully regulated by two ion channels: highly selective cation channels (HSC/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 epithelial permeability. All animal protocols were approved by Emory’s IACUC.

We introduced pathogen toxins via intratracheal instillation or intraperitoneal injections to ASIC1 KO (KO) and wildtype (WT) mouse models. We determined AFC through changes in the concentrations of Evans Blue (EB) or Fluorescein isothiocyanate–dextran (FITC-dextran) in instillates. Permeability of capillaries and alveolar epithelium were investigated by looking at dye accumulation in pulmonary tissue. Concentrations were determined using spectrophotometry. Our findings suggest that LPS and PLY inhibit all HSC-related AFC leaving only NSC-associated AFC. The AFC of LPS and PLY treated KO animals is lower than AFC for LPS treated WT animals. Neither HSC nor NSC are fully functioning or contributing to AFC in toxin treated groups. Observations on permeability are preliminary but suggest fluid accumulation in the interstitium for the ASIC1 KO group. 

Collectively, these results suggest that the regulation of the alveolar fluid layer is dependent on the function of NSC especially after exposure to toxins. HSC and NSC appear to contribute to alveolar permeability and pulmonary capillary integrity but more research is needed. Our findings can help guide treatment for alveolar flooding caused by toxins from different respiratory pathogens by elucidating their mechanism of action. 

Table of Contents

Table of Contents

Introduction...................................................................................................................................... 1

The alveolar fluid layer is critical for respiration.......................................................................... 1

Fluid must be in a steady state across alveolar compartments...................................................... 1

HSC and NSC maintain AFC...................................................................................................... 2

High wet weight-to-dry weight lung ratios suggest fluid accumulation in the interstitium........... 3

HSC and NSC show differing responses to stimuli..................................................................... 3

LPS and PLY have an impact on ENaC/HSC.............................................................................. 4

PLY impairs epithelial and endothelial function........................................................................... 5

Study aims................................................................................................................................... 6

Materials and Methods..................................................................................................................... 7

Animals........................................................................................................................................ 7

Chemicals and reagents................................................................................................................ 7

Intratracheal Instillations.............................................................................................................. 7

Intraperitoneal Injections.............................................................................................................. 8

Alveolar fluid clearance................................................................................................................ 8

Evans blue capillary permeability assay....................................................................................... 9

Wet weight-to-dry weight ratios................................................................................................. 11

Alveolar epithelial permeability.................................................................................................. 11

Single-channel, patch-clamp analysis......................................................................................... 12

Statistical analysis...................................................................................................................... 13

NSC contribute about equally to AFC....................................................................................... 14

LPS and PLY block HSC-associated AFC................................................................................ 15

Acute PLY in alveolar type II cells suppresses HSC activity..................................................... 15

ASIC1 KO mice have more fluid in their lungs......................................................................... 16

Capillary permeability may be impacted by LPS........................................................................ 16

Forty KDa FITC-dextran is a measure of AFC......................................................................... 17

FITC-dextran is a measure of epithelial permeability................................................................. 17

Discussion..................................................................................................................................... 19

NSC are significant contributors to lung fluid balance............................................................... 19

LPS and PLY act on ENaC/HSC to inhibit fluid clearance........................................................ 20

NSC and HSC have contrasting responses to acute PLY.......................................................... 21

Endotoxins may compromise the integrity of epithelial barriers................................................. 22

Summary: NSC are important for proper lung function, especially after endotoxin exposure.... 25

Figures........................................................................................................................................... 26

Figure 1. A schematic diagram of an alveolus............................................................................ 26

Figure 2.  Genetic knockout of nonselective cation channels (NSC) and pharmacological blockage of highly-selective cation channels (HSC) decreases alveolar fluid clearance (AFC)................................................................................... 28

Figure 3. Highly-selective cation channel (HSC)-related fluid clearance is different in male and female wild type mice.   30

Figure 4.  Lipopolysaccharide (LPS) inhibits highly-selective cation channel (HSC)-related alveolar fluid clearance (AFC).        31

Figure 5. Pneumolysin (PLY) inhibits highly-selective cation channel (HSC)-related alveolar fluid clearance (AFC).  AFC of wild type and PLY treated wild type are................................................................................................................... 32

Figure 6. Pneumolysin (PLY) causes a significant increase in the open probability of nonselective cation channels (NSC) on alveolar type 2 cells as assessed by single-patch clamp analysis.................................................................................... 33

Figure 8. ASIC1 KO have fluid accumulation in their interstitium............................................ 35

Figure 9. Forty KDa FITC-dextran detects decreases in fluid clearance caused by endotoxins alveolar fluid clearance (AFC) in wild type mice  37

Figure 10. Different molecular weight FITC-dextran cross the alveolar epithelial to different degrees       38

Figure 11. FITC-dextran is a measure of epithelial permeability................................................ 39

References...................................................................................................................................... 40

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Microbiology Biology, General
Keyword	Alveolar fluid clearance Pneumolysin Nonselective cation channels Lipopolysaccharide
Committee Chair / Thesis Advisor	Douglas C. Eaton, Emory University
Committee Members	Michael Koval, Emory University Nicole Gerardo, Emory University

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