Endothelial cell eNOS regulates sodium excretion in the kidney as determined in endothelial cell-specific eNOS KO and KI mice

A. Lo Bue (Düsseldorf)¹, R. Weins (Duesseldorf)², M. Lindemann (Duesseldorf)², S. K. Heuser (Düsseldorf)³, D. Arifaj (Duesseldorf)², J. Li (Duesseldorf)⁴, J. Stegbauer (Duesseldorf)², M. Cortese-Krott (Düsseldorf)^3
1Myocardial Infarction Research Laboratory Department of Cardiology, Pulmunology and Angiology, Medical Faculty, Heinrich Heine University Düsseldorf, Deutschland; ²Experimentelle Nephrologie Department of Nephrology, Medical Faculty, Heinrich Heine University Duesseldorf, Deutschland; ³Universitätsklinikum Düsseldorf Klinik für Kardiologie, Pneumologie und Angiologie Düsseldorf, Deutschland; ⁴Myocardial Infarction Research Laboratory epartment of Cardiology, Pulmunology and Angiology, Medical Faculty, Heinrich Heine University Duesseldorf, Deutschland

Background: The kidney contributes to blood pressure control by water and sodium handling. Nitric oxide (NO) is a crucial regulator of medullary blood flow and natriuresis through a vasculo-tubular crosstalk. NO is produced by eNOS or nNOS in multiple cell types including endothelial cells (ECs) and epithelial cells. The specific role of EC eNOS in vascular endothelial crosstalk is unknown.
Purpose: The aim of the study is to identify the specific role of EC eNOS to control renal function.
Methods: We compared EC-specific eNOS knockout (KO) mice and their WT littermates (eNOS^flox/floxCdh5-Cre/ERT2^pos/neg, EC eNOS KO/WT), and EC-specific eNOS knockin (KI) mice and their global eNOS KO (gKO) littermates (eNOS^inv/invChd5-Cre/ERT2^pos/neg, EC eNOS KI/gKO). We determined eNOS expression in kidney by real time RT-PCR and Western blotting. Systemic hemodynamics were assessed invasively in anesthetised mice by Millar catheter and in awake mice by radiotelemetry. Renal vascular function was assessed ex vivo in isolated perfused kidneys (IPK) in response to carbachol and angiotensin II (AngII). Renal function was determined in vivo at baseline and after treatment with AngII by analysing urinary sodium excretion at basal conditions and after volume/sodium challenge, urinary albumin/creatinine concentration, and glomerular filtration rate (GFR).
Results: Both EC eNOS KO and gKO mice were hypertensive as compared to WT, while reactivation of eNOS in EC eNOS KI mice rescued the phenotype. Treatment with AngII increased blood pressure in WT, but not in EC eNOS KO mice. As expected, EC eNOS KO and gKO mice lacked carbachol-induced vasodilation in IPK, which was fully restored in EC eNOS KI mice. Acute administration of AngII did not affect vasopressor response in both EC eNOS KO (vs. WT) and EC eNOS KI (vs. gKO). In vivo AngII treatment decreased sodium excretion in WT mice. Interestingly, EC eNOS KO mice showed impaired sodium excretion at baseline compared to WT mice, while AngII did not further decrease it. In gKO mice AngII treatment decreased sodium excretion compared to baseline. Vice versa EC eNOS KI mice showed a preserved sodium excretion after AngII, indicating a protective effect of EC eNOS against AngII-induced impairment. WT mice treated with AngII showed a GFR compensatory increase, while, importantly, EC eNOS KO mice lacked this compensatory response. gKO mice showed decreased GFR after AngII infusion. Reactivation of eNOS in ECs fully rescued the GFR in EC eNOS KI, demonstrating a key role of EC eNOS in regulating GFR. At baseline, EC eNOS KO mice had the same urinary albumin/creatinine ratio as WT controls. Instead, gKO mice showed an increased urinary albumin/creatinine ratio compared to WT, which was fully restored in EC eNOS KI mice.
Conclusion: In the kidney, EC eNOS controls renal vascular responsiveness ex vivo, modulates sodium and urine excretion, and regulates GFR and albumin permeability, thus contributing to systemic hemodynamics and blood pressure control.