Thrombospondin-4 Levels and Peripheral Arterial Disease in Hemodialysis Patients

Study Population and Data Source This is a cross-sectional, single-center study which will be conducted in the Dialysis Center of Tungs' Taichung MetroHarbor Hospital (TTMHH) in the coastal region of central Taiwan. A cohort of 450 patients aged 20 or over, who have been on HD for at least 3 months prior to enrollment (Dec 1, 2021) will be included. The medical charts of these patients are reviewed for eligibility identification, and should be compatible with the inclusion/exclusion criteria and enrolled in our analysis.

In this study, the diagnostic criterion for asymptomatic PAD is an ABI value lower than or equal to 0.9 with no clinical symptoms in the lower limb such as muscle discomfort or intermittent claudication. PAD is considered symptomatic if patients have an ABI ≤0.9 and clinical symptoms or if they undergo previous surgical revascularization procedures or limb amputation. The characteristics of patients will be exclude from our study were (1) baseline ABI values > 1.3 , (2) symptomatic PAD , (3) decompensated cirrhosis , (4) neoplastic diseases , (5) incomplete data, (6) receiving hemodialysis < 3 months and active infection. The baseline data such as demographics, comorbidities, anthropometrics, and relevant laboratory data, clinical diagnosis of PAD based on measurements of ABI, and medication history will be collected.

Ankle Brachial Index Measurements The ABI was measured by trained technicians using the Fukuda Vascular Screening System (VaSera VS-1000™, Fukuda Denshi Co., Ltd., Tokyo, Japan), which measures blood pressure from bilateral arm and ankle (brachial and posterior tibial arteries, resp.) simultaneously by an oscillometric method. The systolic pressure of the arm without dialysis access and the lower value of the ankle systolic pressure were used for the calculation. ABI was calculated by the ratio of the ankle systolic pressure divided by the arm systolic pressure. Of the two ABI values, respectively, calculated from the left- and right-limb measurements, the lowest value is used in this study. All participants were annually measured in a supine position after resting for at least 15 minutes and before dialysis.

In this study, ABI less than 0.90 was considered as evidence of PAD . Absence of PAD was defined as ABI between 0.90 and 1.30 . Individuals with ABI greater than 1.30 were excluded, because this indicates poorly compressible leg arteries and inability to gauge arterial perfusion accurately .

Laboratory Mesaurements Blood was drawn with EDTA anticoagulant in the morning after an overnight fast of at least 12 h before a dialysis session. The samples were separated via centrifugation (4000 rpm, 10 min) and immediately stored at -80°C for subsequent assays. Glucose, high-density lipoprotein cholesterol HDL), triglycerides and total cholesterol are all measured by standard assays using the analyzer. Low-density lipoprotein cholesterol (LDL) is measured by standard assay. Beta 2 microglobulin, high-sensitivity C-reactive protein (hsCRP) and are measuredusing standard nephelometry. Serum hs-cTnT was measured using a sandwich immunoassay method, a novel highly sensitive assay with a lower measurable limit of 3 ng/L. N-terminal probrain natriuretic peptide(NT-proBNP) is measured using a sandwich immunoassay method, the lower limits of quantification were 5 pg/mL. The serum levels of a-Klotho were measured using an enzyme linked immunosorbent assay (ELISA) system, and this assay detects circulating a-Klotho using 2 monoclonal antibodies that specifically recognize the extracellular domain of Klotho, the lower limits of quantification were 6.15 pg/mL and the intra-assay and interassay coefficients of variation of <10%. Plasma blood concentrations of FABP4 were measured by means of (sandwich) ELISA (enzyme-linked immunosorbent assay). Plasma TSP-1 and TSP-4 levels were assessed via ELISA. Calculated intra-assay and interassay coefficient of variation for TSP-4 were 9.3% and 7.7%, respectively.