Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor

The goal of this study was to investigate the viability and synthetic function of rat hepatocytes cocultured with 3T3-J2 fibroblasts in a small-scale microchannel flat-plate bioreactor with and without an internal membrane oxygenator under flow. Bioreactor channel heights ranged between 85 and 500 microm and medium flow rates ranged between 0.06 and 4.18 mL/min. The results showed that the bioreactor without the oxygenator resulted in significantly decreased viability and function of hepatocytes, whereas hepatocytes in the bioreactor with internal membrane oxygenator were able to maintain their viability and function. The shear stress calculations showed that, at lower wall shear stresses (0.01 to 0.33 dyn/cm(2)), hepatocyte functions, measured as albumin and urea synthesis rates, were as much as 2.6- and 1.9-fold greater, respectively, than those at higher wall shear stresses (5 to 21 dyn/cm(2)). Stable albumin and urea synthesis rates for 10 days of perfusion were also demonstrated in the bioreactor with internal membrane oxygenator. These results are relevant in the design of hepatocyte bioreactors and the eventual scaling-up to clinical devices.     

Radial flow hepatocyte bioreactor using stacked microfabricated grooved substrates

Bioartificial liver (BAL) devices with fully functioning hepatocytes have the potential to provide temporary hepatic support for patients with liver failure. The goal of this study was to optimize the flow environment for the cultured hepatocytes in a stacked substrate, radial flow bioreactor. Photolithographic techniques were used to microfabricate concentric grooves onto the underlying glass substrates. The microgrooves served to protect the seeded hepatocytes from the high shear stresses caused by the volumetric flow rates necessary for adequate convective oxygen delivery. Finite element analysis was used to analyze the shear stresses and oxygen concentrations in the bioreactor. By employing high volumetric flow rates, sufficient oxygen supply to the hepatocytes was possible without an integrated oxygen permeable membrane. To implement this concept, 18 microgrooved glass substrates, seeded with rat hepatocytes cocultured with 3T3-J2 fibroblasts, were stacked in the bioreactor, creating a channel height of 100 microm between each substrate. In this bioreactor configuration, liver-specific functions (i.e., albumin and urea synthesis rates) of the hepatocytes remained stable over 5 days of perfusion, and were significantly increased compared to those in the radial flow bioreactor with stacked substrates without microgrooves. This study suggests that this radial flow bioreactor with stacked microgrooved substrates is scalable and may have potential as a BAL device in the treatment of liver failure.     

Microtopography and flow modulate the direction of endothelial cell migration

The migration of vascular endothelial cells under flow can be modulated by the addition of chemical or mechanical stimuli. The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 microm in width at a constant depth of 1 microm. During a 4-h time-lapse observation in the absence of flow, the majority of the prealigned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/cm(2)), irrespective of groove direction with respect to flow. After 4-h exposure to high shear stress (58 dyn/cm(2)) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the migration direction of the cells under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow.     

Computational fluid dynamics modeling of steady-state momentum and mass transport in a bioreactor for cartilage tissue engineering

Computational fluid dynamics (CFD) models to quantify momentum and mass transport under conditions of tissue growth will aid bioreactor design for development of tissue-engineered cartilage constructs. Fluent CFD models are used to calculate flow fields, shear stresses, and oxygen profiles around nonporous constructs simulating cartilage development in our concentric cylinder bioreactor. The shear stress distribution ranges from 1.5 to 12 dyn/cm(2) across the construct surfaces exposed to fluid flow and varies little with the relative number or placement of constructs in the bioreactor. Approximately 80% of the construct surface exposed to flow experiences shear stresses between 1.5 and 4 dyn/cm(2), validating the assumption that the concentric cylinder bioreactor provides a relatively homogeneous hydrodynamic environment for construct growth. Species mass transport modeling for oxygen demonstrates that fluid-phase oxygen transport to constructs is uniform. Some O(2) depletion near the down stream edge of constructs is noted with minimum pO(2) values near the constructs of 35 mmHg (23% O(2) saturation). These values are above oxygen concentrations in cartilage in vivo, suggesting that bioreactor oxygen concentrations likely do not affect chondrocyte growth. Scale-up studies demonstrate the utility and flexibility of CFD models to design and characterize bioreactors for growth of tissue-engineered cartilage.     

Numerical model of fluid flow and oxygen transport in a radial-flow microchannel containing hepatocytes

The incorporation of monolayers of cultured hepatocytes into an extracorporeal perfusion system has become a promising approach for the development of a temporary bioartificial liver (BAL) support system. In this paper we present a numerical investigation of the oxygen tension, shear stress, and pressure drop in a bioreactor for a BAL composed of plasma-perfused chambers containing monolayers of porcine hepatocytes. The chambers consist of microfabricated parallel disks with center-to-edge radial flow. The oxygen uptake rate (OUR), measured in vitro for porcine hepatocytes, was curve-fitted using Michaelis-Menten kinetics for simulation of the oxygen concentration profile. The effect of different parameters that may influence the oxygen transport inside the chambers, such as the plasma flow rate, the chamber height, the initial oxygen tension in the perfused plasma, the OUR, and K(m) was investigated. We found that both the plasma flow rate and the initial oxygen tension may have an important effect upon oxygen transport. Increasing the flow rate and/or the inlet oxygen tension resulted in improved oxygen transport to cells in the radial-flow microchannels, and allowed significantly greater diameter reactor without oxygen limitation to the hepatocytes. In the range investigated in this paper (10 microns < H < 100 microns), and for a constant plasma flow rate, the chamber height, H, had a negligible effect on the oxygen transport to hepatocytes. On the contrary, it strongly affected the mechanical stress on the cells that is also crucial for the successful design of the BAL reactors. A twofold decrease in chamber height from 50 to 25 microns produced approximately a fivefold increase in maximal shear stress at the inlet of the reactor from 2 to 10 dyn/cm2. Further decrease in chamber height resulted in shear stress values that are physiologically unrealistic. Therefore, the channel height needs to be carefully chosen in a BAL design to avoid deleterious hydrodynamic effects on hepatocytes.     

Shear-stress effect on mitochondrial membrane potential and albumin uptake in cultured endothelial cells

Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to shear stress induced by blood flow in vivo, and shear stress affects ATP-dependent macromolecular transport in ECs. However, the relationship between the ATP production and shear stress is still unclear. We, therefore, evaluated mitochondrial ATP synthesis activity in cultured endothelial cells exposed to shear stress, using a confocal laser scanning microscope (CLSM) and a mitochondrial membrane potential probe (5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethyl-benzimidazolycarbocyanine iodide, JC-1). Low shear stress (10 dyn/cm(2)) increased mitochondrial membrane potential by 30%. On the contrary, high shear stress (60 dyn/cm(2)) decreased it by 20%. This observation was consistent with the ATP-dependent albumin uptake into endothelial cells. Our results indicate that ATP synthetic activity is related to the albumin uptake into endothelial cells.     

Experimental technique of measuring dynamic fluid shear stress on the aortic surface of the aortic valve leaflet

Aortic valve (AV) calcification is a highly prevalent disease with serious impact on mortality and morbidity. The exact cause and mechanism of the progression of AV calcification is unknown, although mechanical forces have been known to play a role. It is thus important to characterize the mechanical environment of the AV. In the current study, we establish a methodology of measuring shear stresses experienced by the aortic surface of the AV leaflets using an in vitro valve model and adapting the laser Doppler velocimetry (LDV) technique. The valve model was constructed from a fresh porcine aortic valve, which was trimmed and sutured onto a plastic stented ring, and inserted into an idealized three-lobed sinus acrylic chamber. Valve leaflet location was measured by obtaining the location of highest back-scattered LDV laser light intensity. The technique of performing LDV measurements near to biological surfaces as well as the leaflet locating technique was first validated in two phantom flow systems: (1) steady flow within a straight tube with AV leaflet adhered to the wall, and (2) steady flow within the actual valve model. Dynamic shear stresses were then obtained by applying the techniques on the valve model in a physiologic pulsatile flow loop. Results show that aortic surface shear stresses are low during early systole (<5 dyn/cm2) but elevated to its peak during mid to late systole at about 18-20 dyn/cm2. Low magnitude shear stress (<5 dyn/cm2) was observed during early diastole and dissipated to zero over the diastolic duration. Systolic shear stress was observed to elevate only with the formation of sinus vortex flow. The presented technique can also be used on other in vitro valve models such as congenitally geometrically malformed valves, or to investigate effects of hemodynamics on valve shear stress. Shear stress data can be used for further experiments investigating effects of fluid shear stress on valve biology, for conditioning tissue engineered AV, and to validate numerical simulations.     

Design of well and groove microchannel bioreactors for cell culture

Microfluidic bioreactors have been shown valuable for various cellular applications. The use of micro-wells/grooves bioreactors, in which micro-topographical features are used to protect sensitive cells from the detrimental effects of fluidic shear stress, is a promising approach to culture sensitive cells in these perfusion microsystems. However, such devices exhibit substantially different fluid dynamics and mass transport characteristics compared to conventional planar microchannel reactors. In order to properly design and optimize these systems, fluid and mass transport issues playing a key role in microscale bioreactors should be adequately addressed. The present work is a parametric study of micro-groove/micro-well microchannel bioreactors. Operation conditions and design parameters were theoretically examined via a numerical model. The complex flow pattern obtained at grooves of various depths was studied and the shear protection factor compared to planar microchannels was evaluated. 3D flow simulations were preformed in order to examine the shear protection factor in micro-wells, which were found to have similar attributes as the grooves. The oxygen mass transport problem, which is coupled to the fluid mechanics problem, was solved for various groove geometries and for several cell types, assuming a defined shear stress limitation. It is shown that by optimizing the groove depth, the groove bioreactor may be used to effectively maximize the number of cells cultured within it or to minimize the oxygen gradient existing in such devices. Moreover, for sensitive cells having a high oxygen demand (e.g., hepatocytes) or low endurance to shear (e.g., human embryonic stem cells), results show that the use of grooves is an enabling technology, since under the same physical conditions the cells cannot be cultured for long periods of time in a planar microchannel. In addition to the theoretical model findings, the culture of human foreskin fibroblasts in groove (30 microm depth) and well bioreactors (35 microm depth) was experimentally examined at various flow rates of medium perfusion and compared to cell culture in regular flat microchannels. It was shown that the wells and the grooves enable a one order of magnitude increase in the maximum perfusion rate compared to planar microchannels. Altogether, the study demonstrates that the proper design and use of microgroove/well bioreactors may be highly beneficial for cell culture assays.     

Analysis of cell flux in the parallel plate flow chamber: implications for cell capture studies.

The parallel plate flow chamber provides a controlled environment for determinations of the shear stress at which cells in suspension can bind to endothelial cell monolayers. By decreasing the flow rate of cell-containing media over the monolayer and assessing the number of cells bound at each wall shear stress, the relationship between shear force and binding efficiency can be determined. The rate of binding should depend on the delivery of cells to the surface as well as the intrinsic cell-surface interactions; thus, only if the cell flux to the surface is known can the resulting binding curves be interpreted correctly. We present the development and validation of a mathematical model based on the sedimentation rate and velocity profile in the chamber for the delivery of cells from a flowing suspension to the chamber surface. Our results show that the flux depends on the bulk cell concentration, the distance from the entrance point, and the flow rate of the cell-containing medium. The model was then used in a normalization procedure for experiments in which T cells attach to TNF-alpha-stimulated HUVEC monolayers, showing that a threshold for adhesion occurs at a shear stress of about 3 dyn/cm2.     

Location of scaffolds in bioreactors modulates the hydrodynamic environment experienced by engineered tissues

Physical forces experienced by engineered-tissues during in vitro cultivation influence tissue growth and function. The hydrodynamic environment within bioreactors plays a decisive role in providing the necessary physical stimuli and nutrient transport to support tissue development. Our overall goal is to investigate interrelationships between the local hydrodynamic environment in the bioreactor and the structural and functional tissue properties in order to optimize the production of clinically relevant engineered-tissues. To this end, we used computational fluid dynamics (CFD) modeling to characterize the complex hydrodynamic environment in a wavy-walled bioreactor used for cultivation of tissue-engineered cartilage constructs and examined the changes in the flow field due to the presence of constructs. The flow-induced shear stress range experienced by engineered constructs cultivated in the wavy-walled bioreactor (0-0.67 dyn/cm(2)) was found to be significantly lower than that in the spinner flask (0-1.2 dyn/cm(2)), and to be modulated by the radial or axial position of the constructs. These CFD results are validated by experimental particle-image velocimetry (PIV) measurements previously reported by our group. Results from the present study indicate that the location of constructs in the bioreactor not only affected the magnitude and distribution of the shear stresses on the constructs, but also other hydrodynamic parameters, such as the directional distribution of the fluid velocity and the degree of fluid recirculation, all of which may differentially influence the development of tissue-engineered constructs.     

Endothelial cell response to biomechanical forces under simulated vascular loading conditions

In vivo, endothelial cells (EC) are constantly exposed to the haemodynamic forces (HF) of pressure, wall shear stress and hoop stress. The main aim of this study was to design, create and validate a novel perfusion bioreactor capable of delivering shear stress and intravascular pressure to EC in vitro and to characterise their morphology, orientation and gene expression. Here we report the creation and validation of such a simulator and the dual application of pressure (120/60 mmHg) and low shear stress (5 dyn/cm(2)) to a monolayer of EC established on a non-compliant silicone tube. Under these conditions, EC elongated and realigned obliquely to the direction of applied shear stress in a time-dependent manner. Furthermore, randomly distributed F-actin microfilaments reorganised into long, dense stress fibres crossing the cells in a direction perpendicular to that of flow. Finally, combinatorial biomechanical conditioning of EC induced the expression of the inflammatory-associated E-selectin gene.     

Shear stress-dependent expression of apoptosis-regulating genes in endothelial cells

Laminar shear stress exerts potent anti-apoptotic effects. Therefore, we analyzed the influence of laminar shear stress on the expression of apoptosis-regulating genes in human umbilical vein endothelial cells (HUVEC). Application of high levels of laminar shear stress (15 and 30 dyn/cm(2)) decreased the susceptibility of HUVEC to undergo apoptosis, whereas low shear stress (1 dyn/cm(2)) had no effect. These diminished signs of apoptosis were accompanied by a decreased mRNA expression of apoptosis-inducing Fas receptor. Furthermore, mRNA and protein expression of anti-apoptotic, soluble Fas isoform FasExo6Del and anti-apoptotic Bcl-x(L) were induced. Surprisingly, high shear stress also elevated mRNA and protein expression of pro-apoptotic Bak. The shear stress-induced up-regulation of Bcl-x(L) and Bak mRNA can be abrogated by inhibition of the endothelial NO synthase. We propose that altered expression of Bcl-x(L) and the Fas system is involved in the protective effect of laminar shear stress against apoptosis in human endothelial cells.     

Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats

We hypothesized that aging is characterized by a reduced release of nitric oxide (NO) in response to shear stress in resistance vessels. Mesenteric arterioles and arteries of young (6 mo) and aged (24 mo) male Fischer 344 rats were isolated and cannulated. Shear stress (15 dyn/cm(2))-induced dilation was significantly reduced and shear stress (1, 5, 10, and 15 dyn/cm(2))-induced increases in perfusate nitrite were significantly smaller at all shear stress levels in vessels of aged rats. Inhibition of NO synthesis abolished shear stress-induced release of nitrite. Furthermore, shear stress (15 dyn/cm(2))-induced release of nitrate was significantly higher and total nitrite (nitrite plus nitrate) was significantly lower in vessels of aged rats. Tiron or SOD significantly increased nitrite released from vessels of aged rats, but this was still significantly less than that in young rats. Superoxide production was increased and the activity of SOD was decreased in vessels of aged rats. There were no differences in endothelial NO synthase (eNOS) protein and basal activity or in Cu/Zn-SOD and Mn-SOD proteins in vessels of the two groups, but extracellular SOD was significantly reduced in vessels of aged rats. Maximal release of NO induced by shear stress plus ACh (10(-5) M) was comparable in the two groups, but phospho-eNOS in response to shear stress (15 dyn/cm(2)) was significantly reduced in vessels of aged rats. These data suggest that an increased production of superoxide, a reduced activity of SOD, and an impaired shear stress-induced activation of eNOS are the causes of the decreased shear stress-induced release of NO in vessels of aged rats.     

Transient shear stresses on a suspension cell in turbulence

A model flow field representative of Kolmogorov eddies in turbulence is proposed, and its two parameters are expressed in terms of the known bioreactor variables epsilon, the rate of turbulent power dissipation, and nu, the fluid kinematic viscosity. The trajectory through this flow field of a small sphere representing a cell is determined, and from that the time-varying local shear rate can be found. This allows calculation of the shear stress at any point on the sphere's surface as it rotates in and moves through the model eddy. The maximum shear stress imposed on the cell by the surrounding turbulence has a range of 0.5-5 dyn/cm(2), and can be estimated by 5.33rho(epsilonnu)(1/2). The shear stress has two major frequency components with ranges of 1-4 and 20-80 s(-1); the higher frequency component is estimated by 0.678(epsilon/nu)(1/2). The rotation of the direction of the shear stress vector at each point on the cell's surface is also discussed. Two ways in which external stresses might affect cell growth are proposed.     

Hemodynamics influences vascular peroxynitrite formation: Implication for low-density lipoprotein apo-B-100 nitration

Hemodynamics, specifically, fluid shear stress, modulates the focal nature of atherogenesis. Superoxide anion (O2(-.)) reacts with nitric oxide (.NO) at a rapid diffusion-limited rate to form peroxynitrite (O2(-.) + .NO-->ONOO(-)). Immunohistostaining of human coronary arterial bifurcations or curvatures, where OSS develops, revealed the presence of nitrotyrosine staining, a fingerprint of peroxynitrite; whereas in straight segments, where PSS occurs, nitrotyrosine was absent. We examined vascular nitrative stress in models of oscillatory (OSS) and pulsatile shear stress (PSS). Bovine aortic endothelial cells (BAEC) were exposed to fluid shear stress that simulates arterial blood flow: (1) PSS at a mean shear stress (tau(ave)) of 23 dyn cm(-2) and a temporal gradient (partial differential(tau)/partial differential(t)) at 71 dyn cm(-2) s(-1), and (2) OSS at tau(ave) = 0.02 dyn cm(- 2) and partial differential(tau)/partial differential(t) = +/- 3.0 dyn cm(-2) s(-1) at a frequency of 1 Hz. OSS significantly up-regulated one of the NADPH oxidase subunits (NOx4) expression accompanied with an increase in O2(-.) production. In contrast, PSS up-regulated eNOS expression accompanied with .NO production (total NO(2)(-) and NO(3)(-)). To demonstrate that O2(-.) and .NO are implicated in ONOO(-) formation, we added low-density lipoprotein cholesterol (LDL) to the medium in which BAEC were exposed to the above flow conditions. The medium was analyzed for LDL apo-B-100 nitrotyrosine by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). OSS induced higher levels of 3-nitrotyrosine, dityrosine, and o-hydroxyphenylalanine compared with PSS. In the presence of ONOO(-), specific apo-B-100 tyrosine residues underwent nitration in the alpha and beta helices: alpha-1 (Tyr(144)), alpha-2 (Tyr(2524)), beta-2 (Tyr(3295)), alpha-3 (Tyr(4116)), and beta-2 (Tyr(4211)). Hence, the characteristics of shear stress in the arterial bifurcations influenced the relative production of O2(-.) and .NO with an implication for ONOO(-) formation as evidenced by LDL protein nitration.     

Dynamic contact forces on leukocyte microvilli and their penetration of the endothelial glycocalyx

We develop a theoretical model to examine the combined effect of gravity and microvillus length heterogeneity on tip contact force (F(m)(z)) during free rolling in vitro, including the initiation of L-, P-, and E-selectin tethers and the threshold behavior at low shear. F (m)(z) grows nonlinearly with shear. At shear stress of 1 dyn/cm(2), F(m)(z) is one to two orders of magnitude greater than the 0.1 pN force for gravitational settling without flow. At shear stresses > 0.2 dyn/cm(2) only the longest microvilli contact the substrate; hence at the shear threshold (0.4 dyn/cm(2) for L-selectin), only 5% of microvilli can initiate tethering interaction. The characteristic time for tip contact is surprisingly short, typically 0.1-1 ms. This model is then applied in vivo to explore the free-rolling interaction of leukocyte microvilli with endothelial glycocalyx and the necessary conditions for glycocalyx penetration to initiate cell rolling. The model predicts that for arteriolar capillaries even the longest microvilli cannot initiate rolling, except in regions of low shear or flow reversal. In postcapillary venules, where shear stress is approximately 2 dyn/cm(2), tethering interactions are highly likely, provided that there are some relatively long microvilli. Once tethering is initiated, rolling tends to ensue because F(m)(z) and contact duration will both increase substantially to facilitate glycocalyx penetration by the shorter microvilli.     

Plaque-prone hemodynamics impair endothelial function in pig carotid arteries

Hemodynamic forces play an active role in vascular pathologies, particularly in relation to the localization of atherosclerotic lesions. It has been established that low shear stress combined with cyclic reversal of flow direction (oscillatory shear stress) affects the endothelial cells and may lead to an initiation of plaque development. The aim of the study was to analyze the effect of hemodynamic conditions in arterial segments perfused in vitro in the absence of other stimuli. Left common porcine carotid segments were mounted into an ex vivo arterial support system and perfused for 3 days under unidirectional high and low shear stress (6 +/- 3 and 0.3 +/- 0.1 dyn/cm(2)) and oscillatory shear stress (0.3 +/- 3 dyn/cm(2)). Bradykinin-induced vasorelaxation was drastically decreased in arteries exposed to oscillatory shear stress compared with unidirectional shear stress. Impaired nitric oxide-mediated vasodilation was correlated to changes in both endothelial nitric oxide synthase (eNOS) gene expression and activation in response to bradykinin treatment. This study determined the flow-mediated effects on native tissue perfused with physiologically relevant flows and supports the hypothesis that oscillatory shear stress is a determinant factor in early stages of atherosclerosis. Indeed, oscillatory shear stress induces an endothelial dysfunction, whereas unidirectional shear stress preserves the function of endothelial cells. Endothelial dysfunction is directly mediated by a downregulation of eNOS gene expression and activation; consequently, a decrease of nitric oxide production and/or bioavailability occurs.     

Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase

Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10-20 dyn/cm(2)) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm(2) had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-N(G)-monomethyl arginine (l-NMMA) and l-N(G)-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall.