Apelinergic System: an in‐vitro Investigation of Inflammatory & Oxidative Stress in Chronic Kidney Disease

Abstract

Over 37 million US adults have Chronic Kidney Disease (CKD). Of that population–35% are Black. With 4x higher predisposition to developing renal failure due to comorbidities (i.e. hypertension and diabetes), there is a dire need to address both preventative and therapeutic options in this population. Besides dialysis and transplant as reactionary treatments of CKD, vitamin E—an antioxidant—has been given to those in late stage CKD. Our study aims to address mechanisms behind CKD pathology. The 5/6 nephrectomy (5/6Nx) rat CKD model provides the opportunity to isolate the effects of impaired renal function without significant confounding comorbidities. A hallmark feature of both rat and human CKD is adverse vascular remodeling which is often preceded by endothelial cell (EC) dysfunction. With respect to EC dysfunction, inflammatory and oxidative imbalances are suspected to be involved. Our previous transcriptomic analysis of kidney tissues from the 5/6Nx rat identified a significant increase in apelin receptor (APLNR) expression compared to sham controls. In addition, the data revealed several pro‐inflammatory cytokines (IL‐6 and IL‐1β) preceding APLNR transcript increases. Treatment of Normal Rat Kidney epithelial (NRK) cells with IL‐6 caused a significant reduction in apelin (APLN) ligand, while IL‐1β augmented APLNR transcript levels. After further study of the 3 pathways, a common downstream target, protein kinase B (AKT), arose. Interestingly enough, AKT is responsible for various vascular cascades including interaction with well‐known reactive oxygen species (ROS) producer–NADPH Oxidase 4 (NOX4). Hydrogen Peroxide (H2O2), a ROS product of NOX4, coordinates vasoactivity through AKT signaling and has overlapping function with apelinergic (APJ) system in the vasculature. Due to the inflammatory and oxidative nature of CKD, we want to address the relationship between H2O2 and the APJ system. We hypothesize that pretreating NRK cells with APLN prior to administration of H2O2 will have 2 effects: oxidant attenuation and antioxidant augmentation. NRK cells were pretreated with 2.5ng/mL apelin or 30mg/mL vitamin E 48‐hours prior to addition of 20mM H2O2. At study termination, cells were collected/quantified using the Bradford protein assay. Protein expression was measured via Western blots against catalase, an antioxidant marker, and GPx, an oxidant protein. One‐Way ANOVA and Tukey Post‐Hoc test were used to establish statistical significance (p<0.05). Catalase: no significant expression changes after APLN or vitamin E treat (p = 0.10), but significant increases after H2O2 treat (p = 0.05) compared to control. GPx: significant increases in expression after APLN treat compared to [control (p=0.0002), vitamin E (p=0.04), and H2O2 + APLN (p=0.0001)] and vitamin E compared to H2O2 + Vitamin E treat (p=0.01). Data presented as mean ± SEM (n=3). These studies assessed functionality of the APJ system in an in‐vitro model of CKD. Both of the inflammatory cytokines are regulators of APJ system transcription, which is relevant for patients in a chronic inflammatory state. These studies also highlight the interaction between APJ system and oxidative stressors. Results here begin to lay the foundation for future studies involving the APJ system as a viable target for assessing CKD pathology.