Laminar flow substantially affects the morphology and functional phenotype of glomerular endothelial cells.

Daan C ‘T Hart,Johan Van Der Vlag,Tom Nijenhuis,Nicole Endlich

doi:10.1371/journal.pone.0251129

Abstract

Shear stress induced by laminar blood flow has a profound effect on the morphology and functional phenotype of macrovascular endothelial cells. The influence of laminar flow on the glomerular microvascular endothelium, however, remains largely elusive. The glomerular endothelium, including its glycocalyx, is a crucial part of the glomerular filtration barrier, which is involved in blood filtration. We therefore investigated the influence of laminar flow-induced shear stress on the glomerular endothelium. Conditionally immortalized mouse glomerular endothelial cells were cultured for 7 days under a laminar flow of 5 dyn/cm2 to mimic the glomerular blood flow. The cells were subsequently analysed for changes in morphology, expression of shear stress-responsive genes, nitric oxide production, glycocalyx composition, expression of anti-oxidant genes and the inflammatory response. Culture under laminar flow resulted in cytoskeletal rearrangement and cell alignment compared to static conditions. Moreover, production of nitric oxide was increased and the expression of the main functional component of the glycocalyx, Heparan Sulfate, was enhanced in response to shear stress. Furthermore, glomerular endothelial cells demonstrated a quiescent phenotype under flow, characterized by a decreased expression of the pro-inflammatory gene ICAM-1 and increased expression of the anti-oxidant enzymes HO-1 and NQO1. Upon exposure to the inflammatory stimulus TNFα, however, glomerular endothelial cells cultured under laminar flow showed an enhanced inflammatory response. In conclusion, laminar flow extensively affects the morphology and functional phenotype of glomerular endothelial cells in culture. Furthermore, glomerular endothelial cells respond differently to shear stress compared to macrovascular endothelium. To improve the translation of future in vitro studies with glomerular endothelial cells to the in vivo situation, it appears therefore crucial to culture glomerular endothelial cells under physiological flow conditions.

Highlights

The nephron is the functional unit of the kidney, in which the glomerulus is responsible for filtration of the blood, a process in which passage of plasma proteins into the urine is restricted in a charge- and size selective manner [1]
We examined the effect of flow on the expression of Kruppel-Like Factor 2 (KLF2) and CYP1B1 in glomerular endothelial cells (GEnC), and found that the expression of both KLF2 and CYP1B1 was enhanced in GEnC cultured under flow compared to GEnC cultured under static conditions (p
We demonstrated altered morphology of GEnC, increased expression of shear stress-responsive genes, elevated nitric oxide (NO) production, and an altered anti-oxidant and inflammatory response

Summary

Introduction

The nephron is the functional unit of the kidney, in which the glomerulus is responsible for filtration of the blood, a process in which passage of plasma proteins into the urine is restricted in a charge- and size selective manner [1]. The endothelial glycocalyx plays an important role in glomerular function, by contributing to endothelial barrier function, glomerular structural integrity and by preventing immune cell and cytokine adhesion [3,4,5]. Hyaluronic Acid (HA) is the main non-sulfated glycosaminoglycan (GAG) in the glycocalyx, providing its gel-like structure. Heparan Sulfate (HS) is the main functional sulphated GAG contributing to the charge-selective function of the endothelial barrier. HS degradation compromises the barrier function of the endothelium, and the glycocalyx transforms into a proinflammatory docking station that facilitates binding of cytokines [3,5,6,7]. Podocytes are highly specialized epithelial cells that wrap around the glomerular capillary with their foot processes, forming a sieve-like structure between adjacent foot processes, called the slit diaphragm

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