Study Design: This was a cross-sectional study of children and adolescents with pSLE who were prospectively recruited to undergo comprehensive non-invasive cardiovascular testing from October 2018 to June 2019.
Study Population: Subjects ages 9-21 years inclusive, meeting American College of Rheumatology or Systemic Lupus International Collaborating Clinics classification criteria for SLE by age 18, were recruited from the pediatric rheumatology clinic at a tertiary academic center. We excluded subjects with chronic kidney disease stage 3 or greater (estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2 for ≥3 months), on dialysis, after kidney transplantation, or with a history of obstructive sleep apnea, which are known to be independently associated with nocturnal hypertension. We also excluded subjects with known hypertension at enrollment, but patients with a history of resolved hypertension diagnosis were eligible for inclusion.
Blood Pressure Outcomes:
Casual BP assessment: Trained research nurses measured casual BP using the auscultatory method at least twice in the right upper extremity while in the seated position with an aneroid sphygmomanometer (Mabis MedicKit 5; Mabis Healthcare, Waukegan, IL). If there was a discrepancy of more than 5 mmHg between two measurements in either systolic (SBP) or diastolic blood pressure (DBP), a third measurement was taken and the average of the three measurements was recorded.
Ambulatory BP assessment: 24-hour ABPM was performed using oscillometric Spacelab 90217 monitors (Spacelabs Medical, CA) according to American Heart Association guidelines for standard ambulatory assessment. Systolic and diastolic BP were measured every 20 minutes while awake and every 30 minutes during sleep for 24 hours. The primary outcome was the presence of non-dipping BP, defined as < 10 % decrease from mean daytime to mean nighttime systolic or diastolic BP. Secondary outcomes included masked hypertension (normal casual BP with abnormal mean 24-hr BP or BP load), white coat hypertension (elevated casual BP with normal mean 24-hr BP and BP load), nocturnal hypertension (> 25% of nighttime BP measurements above the 95th percentile for age, sex and height), and average 24-hour systolic or diastolic BP load (total percentage of BP values above the 95th percentile).[13,14,19] BP percentiles and ABPM hypertension category were classified based on the updated American Academy of Pediatrics clinical practice guidelines.
Measures of Vascular Function and Stiffness:
Peripheral endothelial function: Endothelial vasomotor function was assessed in the fasting state during morning hours in a temperature controlled vascular lab using the EndoPAT device (Endo-PAT2000, Itamar-Medical, Caesarea, Israel), as previously described. Digital pulse amplitude readings pre- and post-deflation were used to calculate the reactive hyperemia index (lnRHI), defined as the natural log transformation of the ratio of the post-deflation pulse amplitude over the baseline measurement in the hyperemic finger divided by the corresponding ratio in the control finger. LnRHI values ≤ 0.51 are considered abnormal.
Aortic stiffness: Carotid-femoral pulse wave velocity (PWV) was measured using the SphigmoCor Vx system (AtCor Medical Pty Ltd, Australia). PWV standard deviation scores (SDS) for age and sex were calculated using published reference values for mean transit velocity. Pulse wave analysis (PWA) was also performed to calculate the aortic augmentation index, which approximates aortic stiffness.
Structural Measure of Subclinical Atherosclerosis:
Carotid intima-media thickness (IMT): One experienced sonographer performed all high-resolution, real-time B-mode carotid ultrasound studies (ATL 3000). Serial images in longitudinal and transverse planes were obtained in the supine position at a 45° angle of insonation. Using edge-tracking software (EchoPAC PC, GE Medical Systems), an echocardiologist experienced in measuring carotid IMT (S.N.) performed three separate IMT measurements at the start of the R wave on the electrocardiograph (end-diastole) in the far wall of the right and left distal common carotid artery 10 mm proximal to the origin of the carotid bifurcation, the carotid bulb, and the internal carotid artery 10 mm distal to the bifurcation. The mean of the bilateral CCA measurements (CCA-IMT) and the mean of all 6 segments (mean cIMT) were used as the primary outcome measures of intima-media thickness.[24,25] SDS by age and sex for CCA-IMT were calculated by the LMS method using published reference norms.
Traditional cardiovascular risk factors: Subjects completed height and weight measurements for body mass index (BMI), a Physical Activity Questionnaire, and a demographics survey (race, ethnicity, household income, parental education, family history of early cardiovascular disease). Each subject provided a fasting blood sample for measurement of lipids, high-sensitivity C-reactive protein by immunoassay (Roche Diagnostics c311), and lipoprotein A by enzyme-linked immunosorbent assay (Abcam 212165), which have previously been identified as risk factors for increased rates of atherosclerotic progression in SLE.[5,28] Lipoprotein A values > 95th percentile for age and race/ethnicity (NHANES III) were considered abnormal.
Disease-related factors: Additional clinical data was abstracted from the electronic medical record, including disease duration, SLE manifestations, SLE Disease Activity Index (SLEDAI-2K) scores, glucocorticoid dose, immunosuppressive medication history, and the most recent serum creatinine and urine protein/creatinine levels. eGFR was calculated using the revised Schwartz equation for subjects £ 18 years of age and the Chronic Kidney Disease Epidemiology Collaboration equation for subjects > 18 years of age.[31,32] Cumulative disease activity was calculated as a time-averaged mean  using historical abstractions of SLEDAI-2K scores from clinic visits. For subjects who never had hypocomplementemia or anti-dsDNA antibodies as a disease manifestation, the corresponding score component of zero was carried forward. We also retrospectively calculated the proportion of time in a Lupus Low Disease Activity State (LLDAS) to account for glucocorticoid dose.
ABPM and other vascular outcome measures were summarized using standard descriptive statistics such as mean and standard deviation or median and interquartile range for continuous variables, and count and frequency for categorical variables. We used Shapiro Wilk’s test to assess for normality. A pre-specified significance level alpha of 0.10 (two-sided) was used for all analyses.
- To assess convergent validity between the magnitude of nocturnal BP dipping and other measures of vascular health, we used Pearson correlation (𝒓) coefficients. Since it is not known whether systolic or diastolic BP is more clinically relevant in SLE, systolic and diastolic BP dip were considered separately. We identified outliers using leverage plots and Cook’s distance, and performed sensitivity analyses with and without influential outliers with a Cook’s distance > 0.22. In a secondary analysis, we used Spearman rank correlation (𝜌) coefficients to evaluate the relationship between 24-hour BP load and other vascular measures.
- Prevalence of non-dipping and secondary ambulatory BP outcomes was calculated using the total number of adequate ABPM studies as the offset. To identify potential risk factors for non-dipping, differences in clinical characteristics by non-dipping status were assessed using Fisher’s exact, Student’s t, or Wilcoxon rank sum tests as appropriate.
- To determine whether non-dipping is associated with atherosclerotic risk, we used two-sample t-tests to test differences in CCA-IMT and mean cIMT between subjects with normal nocturnal dipping and those with non-dipping. To explore the discriminative ability of non-dipping with respect to high risk CCA-IMT (SDS > 2.0), we assessed concordance using the C-statistic.