Assessment of Vascular Function in Patients With Chronic Kidney Disease

Summary

The degree of vascular dysfunction and contributing physiological mechanisms can be assessed in patients with chronic kidney disease by measuring brachial artery flow-mediated dilation, aortic pulse-wave velocity, and vascular endothelial cell protein expression.

Abstract

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this is only partially explained by traditional CVD risk factors. Vascular dysfunction is an important non-traditional risk factor, characterized by vascular endothelial dysfunction (most commonly assessed as impaired endothelium-dependent dilation [EDD]) and stiffening of the large elastic arteries. While various techniques exist to assess EDD and large elastic artery stiffness, the most commonly used are brachial artery flow-mediated dilation (FMD_BA) and aortic pulse-wave velocity (aPWV), respectively. Both of these noninvasive measures of vascular dysfunction are independent predictors of future cardiovascular events in patients with and without kidney disease. Patients with CKD demonstrate both impaired FMD_BA, and increased aPWV. While the exact mechanisms by which vascular dysfunction develops in CKD are incompletely understood, increased oxidative stress and a subsequent reduction in nitric oxide (NO) bioavailability are important contributors. Cellular changes in oxidative stress can be assessed by collecting vascular endothelial cells from the antecubital vein and measuring protein expression of markers of oxidative stress using immunofluorescence. We provide here a discussion of these methods to measure FMD_BA, aPWV, and vascular endothelial cell protein expression.

Introduction

Chronic kidney disease (CKD) is a major public health concern that has reached epidemic proportions, affecting ~11.5% of the population in the United States alone¹. The risk of cardiovascular death or a cardiovascular event in patients with CKD is significantly increased compared with the general population^2-4. Although patients with CKD exhibit a high prevalence of traditional cardiovascular risk factors, this only explains part of their increased incidence of cardiovascular disease (CVD)⁵. Vascular dysfunction is an important nontraditional cardiovascular risk factor gaining increased recognition in the field of nephrology^6-9

Protocol

This protocol follows the guidelines of the Colorado Multiple Institutional Review Board (COMIRB).

1. Preparation for Testing Session

1. Participants should follow these restrictions for most accurate measurements: 12 hr fast from food and caffeine, 12 hr restraint from exercise, 12 hr restraint from smoking, if applicable, >4 half-life restraint from medications if possible (may not be feasible in a population such as CKD patients), and pre-menopausal women should be tested in days 1-7 of the menstrual cycle to minimize hormonal influences.
2. Prepare 500 ml of the dissociation buffer by adding 2 ml of 0.5 M ethy....

Results

FMD_BA is quantified as the peak change in diameter of the brachial artery following reactive hyperemia. Thus, the diameter at rest is compared to the diameter following the end of a 5 min blood pressure cuff occlusion period (Figure 1). Panel A shows a representative ultrasound image of the brachial artery, and Panel B displays a graph of the R-wave gated change in diameter from cuff release to 2 min following, as obtained using commercially available software. As the change is often quite min.......

Discussion

Obtaining accurate results for FMD_BA and aPWV requires acquiring high quality ultrasound images and pressure waveforms, respectively. Central to this is appropriate and continued training and use of each technique by the operator⁴⁴. In addition, it is critical to control for as many external variables that may influence the results as possible by standardizing the testing session (e.g., prior 12 hr fast, climate controlled room, etc.)^44,45. As mentioned above, the u.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
J-wire	St. Jude	404584	2 per collection
Disposable shorts (MediShorts)	Quick Medical	4507
Non-invasive hemodynamic workstation (NIHem)	Cardiovascualr Engineering	N/A	Includes custom ruler.  An alternate system is the Sphygmocor
Ultrasound	G.E.	Model: Vivid7 Dimension	We use a G.E., but there are many companies and models
Vascular software (Vascular Imager)	Medical Imaging Applications	N/A
R-wave trigger box	Medical Imaging Applications	N/A	custom made
Rapid Cuff Inflation System	Hokanson	Model: Hokanson E20
Forearm blood pressure cuff	Hokanson	N/A	custom cuff with 6.5 x 34 cm bladder
HUVECs	Invitrogren	C-015-5C
Donkey serum	Jackson	017-000-121
Pap pen	Research Products International	195505
VE Cadherin	Abcam	ab33168
AF568	Life Technologies	A11011	depends on specifications of microscpe
AF488	Life Technologies	A11034	depends on specifications of microscpe
Nitrotyrosine antibody	Abcam	ab7048
NADPH oxidase antibody	Upstate	07-001
DAPI	Vector	H-1200
Delicate task wipe (Kimwipe)	Fisher Scientific	06-666-A
Plastic paraffin film (parafilm)	Fisher Scientific	13-374-10
Confocal microscope	Olympus	Model: FV1000 FCS/RICS	many options exist