Metabolomic Kidney Input and Output Analyses in Salt‐Sensitive Hypertension

Abstract

RationaleSalt‐sensitive hypertension is globally widespread and associated with increased risk of cardiovascular eventsand kidney disease. Although excess dietary salt intake is a major cause of these damaging effects, many of the underlying mechanisms remain poorly defined. Specifically, it is not understood how the critical energy needs of the kidney are met in face of constantly changing environmental nutrients and how this affects metabolic homeostasis in the kidney.MethodsWe hypothesized that a high salt diet in normal salt‐insensitive Sprague Dawley rat would lead to alterations of renal tubular bioenergetics and substrate metabolism. To test this, metabolomic analysis of arterial plasma (input) and renal venous plasma (filtered output) obtained from unanesthetized instrumented rats was performed using tandem mass spectrometry for comparison on a Q‐Exactive Orbitrap mass spectrometer coupled to a dual‐channel Vanquish Ultra‐Performance Liquid Chromatography system. Plasma metabolites were extracted from 20 µL of plasma from SD rats consuming low salt (0.4% NaCl) or high salt (4% NaCl) for 7, 14, or 21 days. These samples were then analyzed with separation over a hydrophobic (C18) and hydrophilic (HILIC) column under both positive and negative polarity to maximize identification of metabolites identified. Varying amounts of quality control plasma sample pools were used to determine injection sample concentration to analyze for each sample and remaining pools were used for normalization of the runs over time.ResultsA total of 4,603 metabolite features were detected, with 1,205 being identified named compounds. After applying further stringency filters to these named metabolites, 350 were significantly different in response to the varying high salt diets (p <0.05; Benjamini‐Hochberg correction) in at least one of the group comparisons performed (versus low salt and artery versus vein). Differential metabolites include numerous lipids, amino acids, and bioenergetic compounds, among others. There appeared to be a shift in these differential compounds between 14 and 21 days of high salt diet, suggesting a potential metabolic shift between initial responses and prolonged high salt responses. Complementary studies are also underway to compare urine metabolite flux, outer medulla, and cortex in conjunction with the plasma analysis to produce a complete computational prediction model.ConclusionTogether, implementation of this methodology will provide key insights and be valuable in further predicting the emergent properties of complex metabolic interactions in the kidney of rats during the development of salt‐induced hypertension.