SALT-2 study: Role of endothelial surface layer in regulating the sodium balance and extracellular fluid volume

Sodium (Na+) plays a key role in maintaining volume homeostasis and blood pressure (BP). The difference between Na+ intake and excretion, the Na+ balance, is regulated by the kidney. Regulation of the Na+ balance by the kidney is believed to be the main determinant of extracellular fluid volume (ECV). Recent studies have revealed that the Na+ balance is not only regulated by the kidney, but also in the interstitium of the skin. Here, binding of Na+ to glycosaminoglycans (GAGs) allows non-osmotic handling of Na+, thereby acting as a Na+ buffer. Based on these findings, we hypothesize that the endothelial surface layer (ESL), representing a complex sugar layer principally composed of negative-charged GAGs lining the endothelium, is an important determinant of volume homeostasis and BP by its ability to act as an immediate non-osmotic Na+ buffer. A perturbed ESL might therefore lead to an increase in ECV and BP after a salt load. The volume of the ESL varies highly between individuals (0.5-2.3 L) and is known to be smaller in specific patient groups such as diabetic patients and patients with chronic kidney disease. The putative non-osmotic buffer capacity of the endothelial GAGs without commensurate water retention has only been limitedly studied yet, but seems particularly relevant in clinical conditions characterized by volume overload (e.g., heart failure, hypertension, chronic kidney disease). If the endothelial GAGs are involved in non-osmotic Na+ storage, treatment strategies directed to restoration of the ESL would lead to improved BP and ECV control and, conceivably, to better cardiovascular outcome. This study focuses on a novel function of the ESL, namely the capacity to store Na+ non-osmotically.



Objective of the study:
In this study we will identify the role of the endothelial GAGs in Na+ and volume homeostasis. Is there a link between the ESL and individual susceptibility to Na+-excess?



Study design:
In this project, we plan to conduct an experimental interventional cross-over study to investigate the Na+ storing capacity of the endothelial GAGs. For this, the effect of different Na+ conditions on ESL, ECV and BP, will be studied in diabetic and hereditary multiple exostosis (HME) patients.



Study population:
Patients are 12 non-smoking male type 1 diabetes patients (DM1) with microalbuminuria (model for acquired loss of ESL) and 12 male patients with HME (model for genetic loss of ESL through a defect in GAG polymerization).



Intervention:
High salt diet (>200 mmol Na+ daily) for 1 week, and low salt diet (<50 mmol Na+ daily) for one week, each in random order. Furthermore, all subjects will receive a hypertonic salt infusion at day 8 of the low salt diet to study the effects of an acute salt load.



Primary study parameters/outcome of the study:
We will primarily study the effects of a salt load on the haemodynamics, ECV and ESL in patients with acquired and congenital loss of the ESL. Primary endpoint will be the ECV as represented by body weight, BP and iohexol measurements.



Secundary study parameters/outcome of the study:
Other study paramaters consist of indirect measurements of the ESL dynamics and function assessed by imaging of the sublingual microvasculature, measurement of ESL shedding products and determination of the transcapillary escape rate (TER) of I125-albumine. The kidney function will be studied by estimating the glomerular filtration rate (eGFR) and measurement of the fractional Na+ excretion and albuminuria. Finally, skin biopsies will allow studying the role of interstitial GAGs and macrophage influx in response to a salt load.

Until recently, it was believed that the sodium (Na+) balance is only regulated by the kidney. Recent studies have revealed that the Na+ balance is not only regulated by the kidney, but also in the interstitium of the skin. Here, binding of Na+ to glycosaminoglycans (GAGs) allows non-osmotic handling of Na+, thereby acting as a Na+ buffer. Based on these findings, the hypothesis will be that the endothelial surface layer (ESL) representing a complex sugar layer principally composed of negative-charged GAGs lining the endothelium is an important determinant of volume homeostasis and BP by its ability to act as an immediate non-osmotic Na+ buffer. Based on the hypothesis that a perturbed ESL might lead to an increased ECV and BP response after a salt load, this study focuses on a novel function of the ESL, namely the capacity to store Na+ non-osmotically. If the ESL is involved in non-osmotic Na+ storage, treatment strategies directed to restoration of the ESL would lead to improved BP and ECV control.

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High salt diet (>200 mmol Na+ daily) for 1 week, and low salt diet (<50 mmol Na+ daily) for one week, each in random order. Furthermore, all subjects will receive a hypertonic salt infusion at day 8 of the low salt diet to study the effects of an acute salt load.