Cigarette smoking causes epigenetic changes associated with cardiorenal fibrosis

Abstract

Clinical studies indicate that smoking combustible cigarettes promotes progression of renal and cardiac injury, leading to functional decline in the setting of chronic kidney disease (CKD). However, basic studies using in vivo small animal models that mimic clinical pathology of CKD are lacking. To address this issue, we evaluated renal and cardiac injury progression and functional changes induced by 4 wk of daily combustible cigarette smoke exposure in the 5/6th partial nephrectomy (PNx) CKD model. Molecular evaluations revealed that cigarette smoke significantly (P < 0.05) decreased renal and cardiac expression of the antifibrotic microRNA miR-29b-3 and increased expression of molecular fibrosis markers. In terms of cardiac and renal organ structure and function, exposure to cigarette smoke led to significantly increased systolic blood pressure, cardiac hypertrophy, cardiac and renal fibrosis, and decreased renal function. These data indicate that decreased expression of miR-29b-3p is a novel mechanism wherein cigarette smoke promotes accelerated cardiac and renal tissue injury in CKD. (155 words).

Keywords: combustible cigarettes; fibrosis; microRNA; physiology; tobacco-related disease; toxicology.

Fig. 1.

PNx and smoking induce significant increases in cardiac and renal fibrosis. Representative Masson's trichrome ×20 photomicrographs of cardiac and renal tissue fibrosis (white scale bar equals 25 μm) (A) and quantification of fibrosis by calculating the mean percent fibrotic area using computer-aided morphometry for heart (B) and kidney (C). Means ± SE are shown from measurements in n = 9 ShamA, 8 ShamS, 5 PNxA, and 10 PNxS animals. Data were analyzed with 2-way ANOVA; full results can be found in Table 1. Abbreviations: ShamA, sham-operated room air exposure only (no combustible cigarette exposure); ShamS, sham-operated exposed to 5 cigarettes per day 5 days per week for 4 wk; PNxA, 5/6th partial nephrectomy room air exposure only (no combustible cigarette exposure); PNxS, 5/6th partial nephrectomy exposed to 5 cigarettes per day 5 days per week for 4 wk.

Fig. 2.

Smoking decreases microRNA (miR)-29b-3p and regulates its targets mRNAs in rat cardiac tissue. RT-quantitative (q)PCR based assessment of miR-29b-3p expression (A); collagen 1a1 mRNA (Col1a1, B); collagen 3a1 (Col3a1, C); collagen 4a1 (Col4a1, D); matrix metalloprotease 2 (Mmp2, E); integrin beta 1 (Itgb1, F); fibrillin (Fbn1, G); and elastin (Eln, H). Means ± SE of n = 5 independent samples assessed in triplicate per group. Data were analyzed with 2-way ANOVA; full results can be found in Table 2. Abbreviations: ShamA, sham-operated room air exposure only (no combustible cigarette exposure); ShamS, sham-operated exposed to 5 cigarettes per day 5 days per week for 4 wk; PNxA, 5/6th partial nephrectomy room air exposure only (no combustible cigarette exposure); PNxS, 5/6th partial nephrectomy exposed to 5 cigarettes per day 5 days per week for 4 wk.

Fig. 3.

Smoking decreases miR-29b-3p and regulates its targets mRNAs in rat renal tissue. RT-qPCR based assessment of miR-29b-3p expression (A); collagen 1a1 mRNA (Col1a1, B); collagen 3a1 (Col3a1, C); collagen 4a1 (Col4a1, D); matrix metalloprotease 2 (Mmp2, E); integrin beta 1 (Itgb1, F); fibrillin (Fbn1, G); and elastin (Eln, H). Means ± SE of n = 5 independent samples assessed in triplicate per group. Data were analyzed with 2-way ANOVA; full results can be found in Table 3. Abbreviations: ShamA, sham-operated room air exposure only (no combustible cigarette exposure); ShamS, sham-operated exposed to 5 cigarettes per day 5 days per week for 4 wk; PNxA, 5/6th partial nephrectomy room air exposure only (no combustible cigarette exposure); PNxS, 5/6th partial nephrectomy exposed to 5 cigarettes per day 5 days per week for 4 wk.

Fig. 4.

Smoking and PNx increased systolic blood pressure and urinary cotinine excretion. A: systolic blood pressure (BP) (mmHg) as determined by tail cuff 4 wk after surgery and implementation of smoking exposure. B: mean cotinine concentration measured in urine collected over a 24 h period just prior to organ collection. Means ± SE are shown from measurements in n = 9 ShamA, 8 ShamS, 5 PNxA, and 10 PNxS animals. Data were analyzed with 2-way ANOVA; full results can be found to the right of each graph. Abbreviations: mm HG, millimeters of mercury; ShamA, sham-operated room air exposure only (no combustible cigarette exposure); ShamS, sham-operated exposed to 5 cigarettes per day 5 days per week for 4 wk; PNxA, 5/6th partial nephrectomy room air exposure only (no combustible cigarette exposure); PNxS, 5/6th partial nephrectomy exposed to 5 cigarettes per day 5 days per week for 4 wk.

Fig. 5.

Smoking and PNx have effects on overall cardiac and renal organ weight. Data shown are heart weight (g)-to-body weight (kg) ratio (A), and the left kidney weight (g)-to-body weight (kg) ratio (B) as determined at time of organ collection. Means ± SE are shown from measurements in n = 9 ShamA, 8 ShamS, 5 PNxA, and 10 PNxS animals. Data were analyzed with 2-way ANOVA; full results can be found to the right of each graph. Abbreviations: BW, body weight measured in kg; HW, heart weight measured in g; KW, kidney weight measured in g; ShamA, sham-operated room air exposure only (no combustible cigarette exposure); ShamS, sham-operated exposed to 5 cigarettes per day 5 days per week for 4 wk; PNxA, 5/6th partial nephrectomy room air exposure only (no combustible cigarette exposure); PNxS, 5/6th partial nephrectomy exposed to 5 cigarettes per day 5 days per week for 4 wk.

Fig. 6.

Proteinuria, plasma cystatin C, and plasma creatinine and clearance are significantly worsened with PNx and smoking. All data were determined 4 wk following the start of PNx and smoking regimen. A: urinary protein excretion; B: plasma cystatin C levels; C: level of plasma creatinine; D: creatinine clearance calculated with the following formula: urine creatinine × urine vol (ml)/plasma Cr × 24 h × 60 min. Means ± SE are shown from measurements in n = 9 ShamA, 8 ShamS, 5 PNxA, and 10 PNxS animals. Data were analyzed with 2-way ANOVA, full results can be found in Table 6. Abbreviations: BW, body weight; CrCl, Creatinine clearance; ShamA, sham-operated room air exposure only (no combustible cigarette exposure); ShamS, sham-operated exposed to 5 cigarettes per day 5 days per week for 4 wk; PNxA, 5/6th partial nephrectomy room air exposure only (no combustible cigarette exposure); PNxS, 5/6th partial nephrectomy exposed to 5 cigarettes per day 5 days per week for 4 wk.