Cardioprotective Role for Paraoxonase-1 in Chronic Kidney Disease

Abstract

Paraoxonase-1 (PON-1) is a hydrolytic enzyme associated with HDL, contributing to its anti-inflammatory, antioxidant, and anti-atherogenic properties. Deficiencies in PON-1 activity result in oxidative stress and detrimental clinical outcomes in the context of chronic kidney disease (CKD). However, it is unclear if a decrease in PON-1 activity is mechanistically linked to adverse cardiovascular events in CKD. We investigated the hypothesis that PON-1 is cardioprotective in a Dahl salt-sensitive model of hypertensive renal disease. Experiments were performed on control Dahl salt-sensitive rats (SS^Mcwi, hereafter designated SS-WT rats) and mutant PON-1 rats (SS-Pon1^em1Mcwi, hereafter designated SS-PON-1 KO rats) generated using CRISPR gene editing technology. Age-matched 10-week-old SS and SS-PON-1 KO male rats were maintained on high-salt diets (8% NaCl) for five weeks to induce hypertensive renal disease. Echocardiography showed that SS-PON-1 KO rats but not SS-WT rats developed compensated left ventricular hypertrophy after only 4 weeks on the high-salt diet. RT-PCR analysis demonstrated a significant increase in the expression of genes linked to cardiac hypertrophy, inflammation, and fibrosis, as well as a significant decrease in genes essential to left ventricular function in SS-PON-1 KO rats compared to SS-WT rats. A histological examination also revealed a significant increase in cardiac fibrosis and immune cell infiltration in SS-PON-1 KO rats, consistent with their cardiac hypertrophy phenotype. Our data suggest that a loss of PON-1 in the salt-sensitive hypertensive model of CKD leads to increased cardiac inflammation and fibrosis as well as a molecular and functional cardiac phenotype consistent with compensated left ventricular hypertrophy.

Keywords: cardiac hypertrophy; fibrosis; inflammation; left ventricular function; paraoxonase-1.

Figure 1

Echocardiographic evaluation at baseline (on normal chow) shows no differences in left ventricular geometry as well as in global systolic function (N = 8). Data are presented as the mean ± standard error of the mean. Student’s unpaired t-test was used to assess statistically significant differences between the groups. VCF: velocity of circumferential fiber shortening, CI: cardiac index, FS: fractional shortening, LVRWT: left ventricular relative wall thickness, LVEDD: left ventricular end-diastolic dimension, LVESD: left ventricular end-systolic dimension.

Figure 2

Echocardiographic evaluation after 4 weeks on 8% high-salt diet. SS-PON-KO HS rats demonstrated significantly decreased end-diastolic and -systolic dimensions, and a significantly increased relative wall thickness as well as increase in global systolic function as shown by increases in fractional shortening, velocity of fractional shortening, and cardiac index (N = 8). Data are presented as the mean ± standard error of the mean. One-way ANOVA and post hoc multiple comparison tests were used to assess statistically significant differences between the groups. VCF: velocity of circumferential fiber shortening, CI: cardiac index, FS: fractional shortening, LVRWT: left ventricular relative wall thickness, LVEDD: left ventricular end-diastolic dimension, LVESD: left ventricular end-systolic dimension.

Figure 3

Targeted mutation of paraoxonase-1 leads to increased cardiac hypertrophy in a hypertensive renal disease model. (A) SS-PON-1 KO on high-salt diet demonstrate significant increase in heart-weight-to-body-weight ratio compared to SS-WT rats; (B) SS-PON-1 KO on high-salt diet demonstrate significant increase in cardiac hypertrophic gene expression compared to SS-WT rats (N = 8). Data are presented as the mean ± standard error of the mean. One-way ANOVA and post hoc multiple comparison tests were used to assess statistically significant differences between the groups.

Figure 4

Targeted mutation of paraoxonase-1 leads to increased cardiac fibrosis in a hypertensive renal disease model. (A) Collagen area (pixels) (N = 8) and (B) gene expression of Timp-1 in cardiac tissue as assessed by RT-PCR from SS-PON-1 KO and SS-WT rats after 5 weeks of 8% high-salt diet (N = 8). SS-PON-1 KO HS rats display significant increase in cardiac fibrosis, compared to SS-WT HS rats following 5 weeks of 8% high-salt diet. Data are presented as the mean ± standard error of the mean. One-way ANOVA and post hoc multiple comparison tests were used to assess statistically significant differences between the groups.

Figure 5

Targeted mutation of paraoxonase-1 leads to a significant increase in cardiac inflammation in a hypertensive renal disease model. (A) SS-PON-1 KO HS rats demonstrate significant increase in CD68-positive immune cells compared to SS-WT HS rats. (B) SS-PON-1 KO HS rats demonstrate significant elevation in inflammatory gene CCL2 expression as compared to SS-WT HS rats. (C) SS-PON-1 KO HS rats demonstrate significant increase in interstitial immune cell infiltration compared to SS-WT HS rats. Representative H&E histology (left) and quantification (right) (N = 8). Data are presented as the mean ± standard error of the mean. One-way ANOVA and post hoc multiple comparison tests were used to assess statistically significant differences between the groups.

Figure 6

Targeted mutation of paraoxonase-1 contributes to decrease in left ventricular function gene expression in a hypertensive renal disease model. SS-PON-1 KO HS rats showed significant decrease in sarcoplasmic reticulum calcium ATPase 2A (atp2a2) and sodium–calcium exchanger (SLC8A1) gene expression compared to SS-WT rats following 5 weeks of 8% high-salt diet (N = 8). Data are presented as the mean ± standard error of the mean. One-way ANOVA and post hoc multiple comparison tests were used to assess statistically significant differences between the groups.