Increased Levels of ER Stress and Apoptosis in a Sheep Model for Pulmonary Fibrosis Are Alleviated by In Vivo Blockade of the KCa3.1 Ion Channel

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease, characterized by progressive damage to the lung tissues. Apoptosis and endoplasmic reticulum stress (ER stress) in type II alveolar epithelial cells (AECs) and lung macrophages have been linked with the development of IPF. Therefore, apoptosis- and ER stress-targeted therapies have drawn attention as potential avenues for treatment of IPF. The calcium-activated potassium ion channel KCa3.1 has been proposed as a potential therapeutic target for fibrotic diseases including IPF. While KCa3.1 is expressed in AECs and macrophages, its influence on ER stress and apoptosis during the disease process is unclear. We utilized a novel sheep model of pulmonary fibrosis to demonstrate that apoptosis and ER stress occur in type II AECs and macrophages in sheep with bleomycin-induced lung fibrosis. Apoptosis in type II AEC and macrophages was identified using the TUNEL method of tagging fragmented nuclear DNA, while ER stress was characterized by increased expression of GRP-78 ER chaperone proteins. We demonstrated that apoptosis and ER stress in type II AECs and macrophages increased significantly 2 weeks after the final bleomycin infusion and remained high for up to 7 weeks post-bleomycin injury. Senicapoc treatment significantly reduced the rates of ER stress in type II AECs and macrophages that were resident in bleomycin-infused lung segments. There were also significant reductions in the rates of apoptosis of type II AECs and macrophages in the lung segments of senicapoc-treated sheep. In vivo blockade of the KCa3.1 ion channel alleviates the ER stress and apoptosis in type II AECs and macrophages, and this effect potentially contributes to the anti-fibrotic effects of senicapoc.

Figure 1

Schematic diagram of bleomycin infusion protocols in sheep (n = 8) and immunohistochemical staining against GRP78 to identify ER stressed type II AECs and macrophages in sheep parenchymal lung tissues. (a) In individual sheep (n = 8), the left caudal lung segment received infusions of bleomycin, while the right caudal lung segment received saline for internal control purposes. (b) Diagram shows the timeline for bleomycin/saline infusions and the tissue collections. These infusions were repeated at day 14 and tissue samples were harvested at day 28. All the animals were kept for 14 days acclimation period (pre-treatment) prior to bleomycin/saline administration. (c) Representative images of GRP78 immuno-stained sections taken from the differentially treated lung segments. Arrows indicate ER stressed type II alveolar epithelial cells (AECs) and macrophages (M). (d, e) Graphs represent the number of ER stressed type II AECs and macrophages in bleomycin- (BLM-) and saline-infused lung segments (n = 8 sheep). The data was taken from twenty representative, non-overlapping fields randomly captured under 40x magnification. Each bar represents the mean ± standard error of the mean. Significance was determined by t-test.  ^∗∗p < 0.001 and  ^∗∗∗p < 0.0001.

Figure 2

Click-iT™ TUNEL Colorimetric assay to identify apoptotic type II AECs and macrophages in sheep parenchymal lung tissues. (a) A representative photomicrograph, captured under 40x magnification for the evaluation. Arrows indicate apoptotic type II alveolar epithelial cells (AECs) and macrophages (M). Graphs represent the number of apoptotic type II AECs and macrophages between bleomycin and saline infusion at the fibrotic stage in sheep lung tissues (n = 8) (b, c). Each bar represents the mean ± standard error of the mean. The tissues were sampled from lung segments at post mortem as indicated in Figures 1(a) and 1(b). Significance was determined by t-test.  ^∗∗p < 0.001 and  ^∗∗∗p < 0.0001.

Figure 3

The effects of senicapoc and pirfenidone on ER stress induced by bleomycin in type II AECs and macrophages. (a) In individual sheep, the left caudal lung segment received infusions of bleomycin, while the right caudal lung segment received the saline. (b) Diagram shows the timeline of the bleomycin administration, treatment protocols (methylcellulose (control), senicapoc, pirfenidone) of the three groups of sheep, and the tissue collection. (c) Representative images from immunohistochemical staining against GRP78 to identify ER stress in type II AECs and macrophages in sheep lung segments treated with either methylcellulose (control), senicapoc, or pirfenidone. Arrows indicate ER stressed type II alveolar epithelial cells (AECs) and macrophages (M). (d, e) Graphs show the rate of ER stress in type II AECs and macrophages between the different treatment groups (n = 10 sheep/group). The data was collected from twenty representative, non-overlapping fields captured at 40x magnification. Each bar represents the mean ± standard error of the mean. Significance was determined by one-way ANOVA and a Tukey's post hoc test to make multiple comparisons test between groups.  ^∗p < 0.05,  ^∗∗p < 0.001, and  ^∗∗∗p < 0.0001.

Figure 4

Effects of senicapoc and pirfenidone treatment on apoptosis induced by bleomycin in type II AECs and macrophages. (a) Representative images from colorimetric TUNEL assays to identify apoptotic type II AECs and macrophages in sheep lung segments treated with either methylcellulose (control), senicapoc, or pirfenidone. Arrows indicate apoptotic type II alveolar epithelial cells (AECs) and macrophages (M). (b, c) Graphs represent the rate of apoptosis in type II AECs and macrophages between the different treatment groups (n = 10 sheep/group). The data was collected from twenty representative, non-overlapping fields captured at 40x magnification. Each bar represents the mean ± standard error of the mean. The group structure and experimental timeline for procedures in this experiment are given in Figures 3(a) and 3(b). Significance was determined by one-way ANOVA and a Tukey's post hoc test to make multiple comparisons test between groups.  ^∗p < 0.05,  ^∗∗p < 0.001, and  ^∗∗∗p < 0.0001.