A Pilot Study Linking Endothelial Injury in Lungs and Kidneys in Chronic Obstructive Pulmonary Disease

Abstract

Rationale: Patients with chronic obstructive pulmonary disease (COPD) frequently have albuminuria (indicative of renal endothelial cell injury) associated with hypoxemia.

Objectives: To determine whether (1) cigarette smoke (CS)-induced pulmonary and renal endothelial cell injury explains the association between albuminuria and COPD, (2) CS-induced albuminuria is linked to increases in the oxidative stress-advanced glycation end products (AGEs) receptor for AGEs (RAGE) pathway, and (3) enalapril (which has antioxidant properties) limits the progression of pulmonary and renal injury by reducing activation of the AGEs-RAGE pathway in endothelial cells in both organs.

Methods: In 26 patients with COPD, 24 ever-smokers without COPD, 32 nonsmokers who underwent a renal biopsy or nephrectomy, and in CS-exposed mice, we assessed pathologic and ultrastructural renal lesions, and measured urinary albumin/creatinine ratios, tissue oxidative stress levels, and AGEs and RAGE levels in pulmonary and renal endothelial cells. The efficacy of enalapril on pulmonary and renal lesions was assessed in CS-exposed mice.

Measurements and main results: Patients with COPD and/or CS-exposed mice had chronic renal injury, increased urinary albumin/creatinine ratios, and increased tissue oxidative stress and AGEs-RAGE levels in pulmonary and renal endothelial cells. Treating mice with enalapril attenuated CS-induced increases in urinary albumin/creatinine ratios, tissue oxidative stress levels, endothelial cell AGEs and RAGE levels, pulmonary and renal cell apoptosis, and the progression of chronic renal and pulmonary lesions.

Conclusions: Patients with COPD and/or CS-exposed mice have pulmonary and renal endothelial cell injury linked to increased endothelial cell AGEs and RAGE levels. Albuminuria could identify patients with COPD in whom angiotensin-converting enzyme inhibitor therapy improves renal and lung function by reducing endothelial injury.

Keywords: COPD; cigarette smoke; comorbidities; endothelium; kidney.

Figure 1.

Patients with chronic obstructive pulmonary disease (COPD) have renal injury. (A) Representative images of trichrome-stained renal sections from patients with COPD, smoker control subjects (SC), and nonsmoker control subjects (NSC) in the nephrectomy cohort. The patient with COPD has hypoperfused glomeruli with a very widened Bowman space and a small shriveled tuft. The dotted yellow lines trace the glomerular perimeters. The insets show glomeruli at a higher magnification. (B) The percentages of glomeruli having glomerular sclerosis in each group. (C) The percentages of the renal interstitium having fibrosis, and renal tubules that were atrophied. (D) The arterial and arteriolar sclerosis score in each group. In D, scores of 1, 2, and 3 represent none or mild, moderate, and severe sclerosis, respectively. Bar graphs show mean + SEM, boxes in box plots show the median values and 25th and 75th percentiles, and error bars show the 10th and 90th percentiles. The dots shown in the graphs represent outliers. Student’s t test (B) or a Mann-Whitney U test (C and D) were used to perform the statistical analyses. *P < 0.05 versus nonsmoker control subjects or the group indicated. In B–D, 5–13 subjects/group were studied.

Figure 2.

Patients with chronic obstructive pulmonary disease (COPD) have repetitive renal endothelial cell injury. (A) Representative electron photomicrographs of glomerular capillary tufts with no, rare, or several double contours (DC) indicated by arrowheads. DC are capillary wall remodeling pathologies indicative of repetitive cycles of endothelial cell injury followed by remodeling. (B) The mean  percentage of subjects having no, rare, or several DC around renal capillaries as assessed using electron microscopy. (C) DC frequency in COPD, smoker control subjects (SC), and nonsmoker control subjects (NSC). Scores of 1, 2, and 3 represent no, rare, and several DC, respectively. Bar graphs show means, boxes in box plots show the median values and 25th and 75th percentiles, and error bars show the 10th and 90th percentiles. The dots shown in the graph represent outliers. Student’s t test (B) or a Mann-Whitney U test (C) were used to analyze the results. *P < 0.05 versus nonsmoker control subjects or the group indicated. In B and C, 12–21 subjects were studied per group.

Figure 3.

Chronic cigarette smoke (CS) exposure increases urinary albumin/creatinine ratios and induces chronic renal and pulmonary lesions in wild-type mice. (A) UACRs measured in urine samples from C57BL/6 wild-type mice. The dot shown in the plot represents an outlier. (B) Images of glomeruli in hematoxylin and eosin–stained renal sections from mice exposed to air for 6 months or mice exposed to CS for 6 months and treated with saline or enalapril 6 days a week beginning at the 12-week time point and continued for the second 12 weeks of the CS exposures. The dotted red lines trace the glomerular perimeters. The images are representative of 3–4 mice/group. The bar graph shows the mean + SEM glomerular major diameters. (C) Electron microscopy images of podocytes from air-exposed mice or CS-exposed mice treated with saline or enalapril as outlined above. The red lines trace the widths of the base of the podocytes. The bar graph shows mean + SEM podocyte widths. Airspace enlargement (D) and small airway fibrosis (E) were quantified in the three experimental groups. Boxes in box plots show the median values and 25th and 75th percentiles, and error bars show the 10th and 90th percentiles. Data were analyzed using Mann-Whitney U test (A, D, and E) or Student’s t test (B and C). In A–C, 3–4 mice were studied per group, and in D and E, 7–16 mice were studied per group. (A–E) *P < 0.05 versus the air-exposed group or versus the group indicated. ECM = extracellular matrix; UACR = urinary albumin/creatinine ratio; Veh = vehicle.

Figure 4.

Chronic cigarette smoke (CS) exposure induces injury to pulmonary and renal endothelial cells (ECs) in mice and increases renal EC advanced glycation end-products (AGEs) and receptor for AGEs (RAGE) staining, which are abrogated by enalapril therapy. C57BL/6 wild-type mice were exposed to air or CS for 24 weeks. In CS-exposed mice, enalapril or vehicle therapy was initiated at the 12-week time point as described in the legend to Figure 3. (A) Terminal deoxynucleotidyl transferase (TUNEL)-positive ECs (identified by staining lung and kidney sections with a green color to detect TUNEL-positive cells and a red color to detect von Willebrand factor–positive ECs) were counted in 3–5 mice/group. (B) Tissue AGEs levels measured in homogenates of lungs and kidneys from 5–8 mice/group. (C) RAGE immunostaining levels in von Willebrand factor–positive ECs in sections of lungs or kidneys from 3–4 mice/group. Bar graphs show mean + SEM, boxes in box plots show the median values and 25th and 75th percentiles, and error bars show the 10th and 90th percentiles. Data were analyzed using Mann-Whitney U test (A and B, right; C, left) or Student’s t test (A, left; C, right). *P ≤ 0.05 versus air-exposed group or the group indicated. Veh = vehicle.

Figure 5.

Enalapril therapy reduces pulmonary inflammation, urinary albumin/creatinine ratios (UACRs), tissue oxidative stress and advanced glycation end-products (AGEs) levels, and endothelial cell (EC) receptor for AGEs (RAGE) staining in the kidneys and lungs of mice exposed acutely to cigarette smoke (CS). C57BL/6 wild-type mice were exposed to air or CS for 4 weeks. In CS-exposed mice, enalapril therapy versus vehicle (6 d/wk) was initiated beginning at the mid-point of the 4-week CS exposures. (A) Total leukocyte counts in bronchoalveolar lavage samples from 5–7 mice/group. (B) UACRs in 10–13 mice/group. (C) Oxidative stress levels measured as tiobarbituric acid reactive substances (TBARS) in homogenates of lungs and kidneys from 5–11 mice/group. (D) Tissue AGEs levels in homogenates of lungs and kidneys from 5–17 mice/group. (E) RAGE immunostaining levels in von Willebrand factor–positive ECs in sections of lungs (left) or kidneys (right) from 3–4 mice/group. Bar graphs show mean + SEM, boxes in box plots show the median values and 25th and 75th percentiles, and error bars show the 10th and 90th percentiles. Data were analyzed using Student’s t test (A and E) or Mann Whitney U test (B–D). (A–E) *P ≤ 0.05 versus air-exposed group or the group indicated. Two-tailed tests were used for all analyses. (E, right) *P = 0.06 was obtained when comparing the enalapril-treated versus the vehicle-treated groups. BAL = bronchoalveolar lavage; Veh = vehicle.

Figure 6.

Advanced glycation end-products (AGEs) and receptor for AGEs (RAGE) levels are increased in pulmonary and renal endothelial cells in patients with chronic obstructive pulmonary disease (COPD). Confocal images of triple-color immunofluorescence staining of sections of lungs (A) or kidneys (glomeruli, in B) from patients with COPD, smoker control subjects (SC), and nonsmoker control subjects (NSC). Endothelial cells in the sections were labeled with a red fluorophore for von Willebrand factor (vWF). Sections were also stained with a green fluorophore for AGEs and gray color for RAGE, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (blue). The merged images show colocalized staining in yellow. White arrows indicate RAGE staining and green arrows indicate AGEs staining in endothelial cells. The far right panels show a section of a lung (A) or a kidney (B) sample from a patient with COPD stained with isotype-matched nonimmune control antibodies. The images shown are representative of 4–5 subjects/group. Rb = rabbit.