Respiratory infections are a common cause of morbidity and mortality in many diseases including Cystic Fibrosis (CF). In spite of current therapies more than 90% of people with CF die from respiratory infections. CF is caused by mutations in the gene for cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel. How defects in this anion channel led to respiratory infections is unclear. To better understand CF lung disease, we produced CF null pigs. CF pigs show host defense defects, similar to humans. We found that CF pigs have an abnormally acidic airway surface liquid (ASL), the thin layer of fluid lining the lung epithelia. Increasing the pH of the ASL in CF pigs rescues the host defense defect. Thus, we hypothesize that infections in CF are due to an acidic ASL pH, in part, by a dysfunctional CFTR, which normally secretes HCO3-. In addition to CFTR, a number of ion channels and transporters have been hypothesized to be involved in HCO3- secretion and H+ secretion. However, which ion channels and transporters primarily contribute to ASL pH is unclear. If the dominant pathways can be identified, the most efficacious pharmacological therapy to increase ASL pH and presumably decrease lung infections can be developed. To accomplish this, we propose the following specific aims: 1) Determine the channels and transporters that secrete HCO3- into the ASL 2) Determine the primary mechanism of H+ secretion into the ASL. To determine which HCO3--secreting and which H+-secreting proteins are most important, we will use small interfering RNA (siRNA) to selectively knock down specific mRNAs and pharmacologic agents to inhibit ion channels and transporters. We will measure the effect of inhibiting these proteins on ASL pH in airway epithelia. We will quantify this effect using three techniques: pH sensitive fluorophores, Ussing chamber electrophysiology, and pH stat. By dispersing a pH sensitive fluorophore into the ASL, we can measure the effects of on ASL pH directly. In Ussing Chambers, we will voltage-clamp cultures to measure electrogenic HCO3- transport generated by pharmacologic stimulus. To account for electro-neutral ion transport, we will use pH stat to pH-clamp and measure rates of H+ or HCO3- secretion. We expect that with these techniques, we will identify which proteins contribute most to HCO3- secretion and H+ secretion into the ASL. We will then test the effects on CF host defense defects in vitro by measuring bacterial killing and ASL viscosity. Knowing which proteins are the major contributors to ASL pH, is the first step towards our long-term goal of developing drugs to increase ASL pH. Using our access to CF pigs, we can test the drugs in vivo, and then develop them as therapies for humans. These drugs would have a major impact for not only CF, but also for other diseases like COPD, which also show acidic ASL and bacterial infections. This project is a direct application of understanding a physiology mechanism to better human health, the mission of NHLBI.

Public Health Relevance

Respiratory infections are a common cause of morbidity and mortality in CF and COPD. We found that when the thin layer of liquid on the surface of airways is more acidic, bacteria have increased survival, possibly leading to the chronic infections found in these diseases. This project aims to ascertain the most relevant proteins in secreting acid and base so that most efficacious drug targets can be determined, which will decrease the abnormal acidity, and consequently reduce bacterial lung infections, increasing quality of life in patients affected by these diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
1F30HL123239-01
Application #
8716459
Study Section
Special Emphasis Panel (ZRG1-F10A-S (20))
Program Officer
Tigno, Xenia
Project Start
2014-05-01
Project End
2018-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
1
Fiscal Year
2014
Total Cost
$28,122
Indirect Cost
Name
University of Iowa
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242