CD14 Deletion Amplifies Dahl Salt‐Sensitive Hypertension and Renal Damage through a NOX2‐dependent Mechanism

Abstract

Previous work has implicated the immune system in potentiating salt‐sensitive (SS) hypertension. The innate immunity protein Cluster of Differentiation 14 (CD14), a co‐receptor with Toll‐like Receptor 4 in the inflammasome pathway of macrophages, has been associated with cardiovascular disease in humans. Interestingly, genetic deletion of CD14 in the Dahl SS rat (SSCD14‐/‐) confers a significant exacerbation of salt‐induced hypertension and associated renal disease that is specific to females. We speculated that CD14 may modulate the production of reaction oxygen species (ROS), since our previous findings implicate NADPH oxidase 2 (NOX2) derived ROS in immune cells in the pathogenesis of salt‐sensitive hypertension. In initial in vitro studies (n>4), peritoneal macrophages from Dahl SS, SSCD14‐/‐, and SSp67phox‐/‐ (a strain lacking the critical p67phox subunit of NOX2) rats were stimulated with phorbol 12‐myristate 13‐acetate, and the production of superoxide was assessed by chemiluminescence via luminol derivative L‐012. Specificity was determined using superoxide dismutase which diminished all detected signal. Superoxide release was 2.4x greater from peritoneal macrophages from female SSCD14‐/‐ when compared to SS macrophages. The SSp67phox‐/‐, lacking NOX2 activity, served as a negative control. The present study tests the hypothesis that the amplification of salt‐sensitive hypertension and renal damage in Dahl SS females lacking CD14 is mediated by NOX2. A double knockout Dahl SS rat was bred to lack both CD14 and functional NOX2 (SSCD14‐/‐p67phox‐/‐ (DKO)) to investigate a potential link between CD14 and oxidative stress. The hypertensive and kidney injury phenotype in response to a 3 week high salt challenge (HS, 4.0% NaCl, AIN‐76A) was examined in DKO females compared to SSCD14‐/‐ and SSp67phox‐/‐ females. After 3 weeks of HS, DKOrats had a significantly blunted change in mean arterial pressure (MAP) from baseline (0.4%) to HS day 21 compared to SSCD14‐/‐ rats (15 mmHg, n=3 versus 38 mmHg, n=7, p=0.01). Importantly, HS MAP in the DKO was not different than that of the SSp67phox‐/‐ (121 ± 4 mmHg versus 120 ± 2 mmHg, n=3, p>0.05), indicating that ROS production from NOX2 is likely mediating the amplified elevation of MAP following CD14 deletion. As an index of renal damage, albuminuria was significantly reduced in the DKO (11.8 ± 1.7 mg/day, n=9, p=0.004) compared to the SSCD14‐/‐ (101.8 ± 17.5 mg/day, n=12) at HS day 21. There was no difference in albuminuria between the DKO and SSp67phox‐/‐(11.7 ± 2.9 mg/day, n=7, p>0.05). Flow cytometric analysis of immune cells isolated from the kidneys of these rats indicated a reduction in renal inflammation in the DKO compared to the SSCD14‐/‐. There were fewer CD45+ total leukocytes (46% reduction), CD3+ T cells (45%), CD45R+ B cells (60%), and CD11b/c+ monocytes/macrophages (46%) in DKO (n=7) versus SSCD14‐/‐ kidneys (n=7). Together, these data suggest a role of CD14 in reducing the pathogenesis of salt‐sensitive hypertension and renal damage in a process dependent upon NOX2‐mediated oxidative stress. Here we highlight a novel renoprotective role of CD14.