We conducted a single-center retrospective cohort study. Patients with T1D who received a first SPKT between 2008 and 2020 were included. Data were collected at transplant and during the first year after SPKT. The study protocol was conducted according to the principles of the Declaration of Helsinki and approved by the institution’s research ethics committee.

All participants were diagnosed with T1D, ascertained by experienced endocrinologists, on the basis of specific pancreas antibodies (glutamic acid decarboxylase and/or tyrosine-phosphatase-like protein IA2 antibodies), abrupt onset of the hyperglycemia, and/or the need for continuous insulin treatment from the beginning. Those with ESKD (CKD stages IV–V [estimated glomerular filtration rate [eGFR] < 20 mL/min/1.73 m2)] received SPKT. Exclusion criteria were type 2 diabetes (T2D) or increased pretransplant C-peptide levels (> 3 ng/mL), previous or active glucocorticoid or immunosuppressive treatment, morbid obesity (body mass index [BMI] ≥ 40 kg/m2), drugs interfering with the HPA axis, or previous solid organ transplant. In particular, all subjects with active or past steroid use were excluded from the study.

All patients followed a standardized multidisciplinary pretransplant evaluation and follow-up, which included the assessment of graft function and the evaluation of pre-existent diabetes complications [24].

To ascertain the association of midnight cortisol on the cardiovascular risk profile and SPKT evolution, only those subjects who had a pretransplant blood extraction between 11:00 pm and 1:00 am were selected. A total of 29 subjects were identified and included.

Demographic and clinical variables such as age, sex, smoking habit, cardiovascular comorbidities, cardioprotective drugs, previous CV disease, and history of diabetes complications were obtained from medical records.

Physical examination included weight and height (calculating the BMI, accordingly), blood pressure measured after a 5-min supine rest, and following 3 min of quiet standing, ankle brachial index (ABI; using a standardized protocol [25]), vibration perception threshold measured in both inferior limbs by a biothesiometer (Bio Medical Instrument Co, Newbury, OH), and exhaustive examination of the feet. All the procedures were performed by trained nurses.

The presence of diabetic retinopathy was always ascertained and graded by an ophthalmologist. Diabetic neuropathy was evaluated by symptoms, an abnormal vibration perception threshold value (≥ 25 V; measured by a biothesiometer bilaterally on the protuberance of the first toes and on the spine of the tibias), or the presence of orthostatic hypotension [a reduction of ≥ 20 mmHg in SBP or ≥ 10 mmHg in diastolic blood pressure (DBP) after 5 min of supine rest and following 3 min of quiet standing]. Peripheral arterial disease was evaluated by symptoms and history of revascularization or amputation, or ABI < 0.9 [26]. Ischemic heart disease was evaluated by previous history of myocardial infarction, angina, history of revascularization or positive stress test, and cerebrovascular disease by previous history of ischemic or hemorrhagic stroke or transient ischemic attack. The complete pretransplant work-up has been thoroughly described previously [5, 27].

Standardized assays were used to measure glucose, HbA1c, lipid profile (including total cholesterol, HDL-cholesterol, triglycerides; LDL-cholesterol [LDL-c] was calculated with the Friedewald formula), C-peptide, and creatinine and urinary albumin-to-creatinine ratio in the local laboratory. The eGFR was obtained with the CKD Epidemiology Collaboration equation (CKD-EPI).

A blood test was performed between 11:00 pm and 1:00 am on the night before SPKT to measure midnight cortisol, cortisol-binding globulin (CBG), and sTWEAK. All blood samples from 2008 till 2020 were measured in the hormonal laboratory in the institution as follows: serum cortisol was measured by chemiluminescence immunoassay (Atellica IM1600, Siemens Healthineers, Tarrytown, NY, USA), CBG was measured by radioimmunoassay (DiaSource, Louvain-la-Neuve, Belgium), and sTWEAK was measured using enzyme-linked immunosorbent assay (kit BMS2006INST, Bender MedSystems, Burlingame, California).

Pancreas graft failure was defined as any of the following: (a) graft removal, (b) C-peptide < 1 ng/mL, (c) total daily insulin dose > 0.5 U/kg, or (d) patient death. Pancreas early graft failure (EGF) was defined as any pancreas graft failure during the first 90 days following SPKT. Kidney graft failure was defined as return to dialysis, retransplantation, or patient death. Kidney delayed graft function was defined as the need for at least one session of hemodialysis during the first week following SPKT.

Cardiovascular events (CVE) in the year following SPKT were registered, including cardiac, cerebrovascular, or peripheral arterial disease, as described above.

Data are presented as median and 25th and 75th percentiles, mean ± SD for non-normal and normal distributions, respectively, or number (percentage). Normal distribution of continuous variables was evaluated with the Kolmogorov–Smirnov test.

The cohort was divided according to MC and sTWEAK quartiles. Then, multiple-group analyses in clinical and laboratory variables were performed using analysis of variance (ANOVA), Kruskal–Wallis, and chi-square tests as appropriate. Bonferroni and Jonckheere–Terpstra tests were performed to assess linear trends, for parametric and non-parametric variables, respectively. The differences between baseline and 1-year follow-up in cardiovascular risk factors were assessed with paired tests (Student’s t test and Wilcoxon test for continuous variables; McNemar’s test for categorical variables) for all the cohort and according to MC quartiles.

To explore for independent relationships between MC (independent variable) and baseline SBP (dependent variable) a logistic binary regression multivariable model was constructed. The model included age, sex, BMI, eGFR, and diabetes duration. A logistic regression analysis model was also constructed to explore for independent relationships between MC (independent variable) and diabetic neuropathy (dependent variable), including age, sex, SBP, sTWEAK, and diabetes duration as co-variables.

Significance level was defined as a p value < 0.05. IBM SPSS Statistics 23.0 (SPSS, Inc; Chicago, Illinois) was used to perform the statistical analysis.

Similar methods have been presented elsewhere [28].