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Supplemental Material

Corresponding to

Diabetic neuropathy is independently associated with worse graft outcomes and incident cardiovascular disease after pancreas transplantation: a

retrospective cohort study in type 1 diabetes

Laura Boswell, MD,^1,2 Pedro Ventura-Aguiar, MD, PhD,^3,4 Aida Alejaldre, MD, PhD,⁵ Judith Navarro-Otano, MD, PhD,⁵ Frederic Cofan, MD, PhD,^3,4 Tonet Serés-Noriega, MD,¹ Adriana Pané, MD,¹ Enrique Montagud-Marrahi, MD,³ Alicia Molina-Andújar, MD,³ Montserrat Ruiz, RN,¹ David Cucchiari, MD, PhD,³ Mireia Musquera, MD, PhD,⁶ Joana Ferrer-Fàbrega, MD, PhD,⁷ Fritz Diekmann, MD,

PhD,^3,4,8 Enric Esmatjes, MD, PhD,^9,10and Antonio J. Amor, MD, PhD¹

1 Diabetes Unit, Endocrinology and Nutrition Department, Hospital Clínic de Barcelona, Barcelona, Spain.

2 Endocrinology and Nutrition Department, Althaia University Health Network, Manres a, Spain

3 Renal Transplant Unit, Nephrology and Kidney Transplantation Department, Hospital Clínic de Barcelona, Barcelona, Spain.

4 Laboratori Experimental

de Nefrologia i Trasplantament (LENIT), Institut d'Investigacions Biomèdiques Augus t Pi i Sunyer (IDIBAPS), Barcelona, Spain.

5 Neurology Department, Hospital Clínic de

Barcelona and Institut d´Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain.

6 Urology Department, Hospital Clinic de Barcelona, Barcelona, Spain.

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7 Hepatobiliopancreatic and Liver Transplant Department, Hospital Clinic de Barcelona, Barcelona, Spain

8 Red de Investigación Renal (REDINREN), Madrid, Spain.

9 Diabetes Unit, Endocrinology and Nutrition Department, Hospital Clínic de Barcelona, Institut d'Investigacions Biomèdiques August

Pi i Sunyer (IDIBAPS), Barcelona, Spain.

10 Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III (ISCIII), Madrid, Spain.

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Supplemental Material and Methods Surgical technique

The surgical technique has previously described.^1-3 All pancreas transplants

procedures were performed with enteric anastomosis. Duodeno-jejunal anastomosis with intraperitoneal position was used until 2016, and from June 2016 onward a retroperitoneal position with duodeno-duodenal anastomosis. Systemic venous drainage was performed through anastomosis between graft’s portal vein to the recipient’s inferior vena cava. For the arterial anastomosis, superior mesenteric artery was end-to-end anastomosed to splenic artery. In the current era, a Y graft is used. Arterial anastomosis is performed to the recipient common iliac artery or directly to the aorta.

Anticoagulation and Immunosuppression

Anticoagulation and immunosuppression have been previously specified.⁴ Briefly, subcutaneous enoxaparin 20 mg bid was started 8h postsurgery and was

maintained until patient discharge (in the absence of thrombotic/haemorrhagic complications), and acetylsalicylic acid 50 mg/day was started at 12h postsurgery until discharge, when it is increased up to 100 mg/day. Regarding

immunosuppression, induction therapy consisted in 2 doses of anti-IL2 monoclonal antibody (basiliximab; 20 mg at day 0 and at day +4 after surgery) until July 2013, and thereafter replaced by rabbit anti-human lymphocytes polyclonal antibodies (either Thymoglobulin 1.25mg/Kg/day or ATG 2.5mg/Kg/day) for 4 consecutive days.

Maintenance immunosuppression protocol was based on triple therapy with calcineurin inhibitor (cyclosporine A until 2005, and thereafter tacrolimus),

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mycophenolate, and steroids (methylprednisolone in the immediate posttransplant period, followed by oral prednisone).

References

1. Gilabert R, Fernández-Cruz L, Real MI, et al.Treatment and outcome of pancreatic venous graft thrombosis after kidney-pancreas transplantation. Br J Surg. 2002;89(3):355–360.

2. Barrufet M, Burrel M, Angeles García-Criado M, et al. Pancreas transplants venous graft thrombosis: endovascular thrombolysis for graft rescue.

Cardiovasc Intervent Radiol. 2014;37(5):1226–1234.

3. Ferrer J, Molina V, Rull R, et al. Trasplante de páncreas: ventajas de la posición retroperitoneal del injerto. Cir Esp. 2017;95(9):513–520.

4. Montagud-Marrahi E, Molina-Andújar A, Pané A, et al. Outcomes of pancreas transplantation in older diabetic patients. BMJ Open Diabetes Res Care.

2020;8(1):e000916.

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Table S1. Pretransplant characteristics of the participants according to transplantation modality (SPK vs PAK).

Data are shown as mean ± standard deviation, median (interquartile range) or number (percentage).

aMissing value n=42

b Missing value n=4

c Missing value n=36

§ Missing value n=23

BMI, body mass index; BP, blood pressure; ACEi, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; PAK, pancreas after kidney; SPK, simultaneous pancreas-kidney.

SPK (n=176)

PAK

(n=11) p-value

Clinical characteristics

Age at pancreas transplant (years) 39.8±7.0 40.8±8.5 0.656

Gender (Male) 124 (70.5) 7 (63.6) 0.736

BMI (Kg/m2) 23.4±3.3 22.7±3.3 0.529

<25 127 (72.2) 8 (72.7)

0.784

25-29.9 42 (23.8) 3 (27.3)

³30 7 (4.0) 0 (0)

Active smokers^a 46 (34.1) 3 (30.0) 0.793

Systolic BP (mmHg) 138±23 129±24 0.289

Diastolic BP (mmHg) 81±14 71±11 0.021

Diabetic retinopathy 171 (97.2) 11 (100) 0.736

Cardiovascular disease 44 (25.0) 3 (27.3) 0.866

Diabetes duration (years) 27.0±6.7 29.3±9.7 0.465 Patients receiving dialysis 138 (78.4) 5 (45.5) 0.022

Laboratory characteristics

Total cholesterol (mg/dL) 187±52 154±19 <0.001 Triglycerides (mg/dL) 119 (85-161) 111 (84-163) 0.969

LDL-cholesterol (mg/dl) 106±36 82±14 <0.001

HDL-cholesterol (mg/dL) 54±18 47±11 0.259

HbA1c (%) 7.8±1.7 7.7±1.9 0.813

HbA1c (mmol/mol) 62±18 61±21 0.813

Treatment characteristics

Intensive insulin therapy^b 111 (64.5) 9 (81.8) 0.335

Insulin dose (U/Kg) 0.58±0.20 0.70±0.20 0.079

Lipid-lowering drugs^c 77 (54.2) 4 (44.4) 0.734

ACEi or ARB use^d 106 (68.4) 6 (66.7) 0.914

Use of 3 or more antihypertensive

drugs^d 68 (43.9) 4 (44.9) 0.973

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Table S2. Clinical characteristics of the participants according to orthostatic hypotension at baseline.

Whole cohort (n=187)

Orthostatic hypotension

p-value No

(n=152) Yes (n=28)

Clinical characteristics

Age at transplant (years) 39.9±7.1 39.5±7.2 40.3±6.3 0.611 Gender (Male) 131 (70.1) 105 (69.1) 19 (67.9) 0.898

BMI (Kg/m2) 23.3±3.3 23.2±3.1 23.8±4.1 0.443

<25 135 (72.2) 111 (73.0) 19 (67.9)

0.126

25-29.9 45 (24.1) 37 (24.3) 6 (21.4)

³30 7 (3.7) 4 (2.6) 3 (10.7)

Active smokers^a 49 (33.8) 40 (33.9) 7 (33.3) 0.960

Systolic BP (mmHg) 137±23 136±22 142±22 0.173

Diastolic BP (mmHg) 81±14 79±13 87±18 0.026

Diabetic retinopathy 182 (97.3) 147 (96.7) 28 (100) 0.330 Cardiovascular disease 47 (25.1) 34 (22.4) 8 (28.6) 0.476 Diabetes duration (years) 27.1±6.9 27.1±6.9 26.1±6.2 0.460 Patients receiving dialysis 143 (76.5) 114 (75.0) 23 (82.1) 0.415

Laboratory characteristics

Total cholesterol (mg/dL) 185±51 183±45 195±76 0.293 Triglycerides (mg/dL) 118 (85-161) 120 (88-168) 104 (85-151) 0.345 LDL-cholesterol (mg/dl) 104±35 104±35 104±39 0.950

HDL-cholesterol (mg/dL) 53±17 53±17 51±17 0.611

HbA1c (%) 7.8±1.7 7.7±1.5 8.6±2.1 0.065

HbA1c (mmol/mol) 62±18 61±17 70±24 0.065

Treatment characteristics

Intensive insulin therapy^b 120 (65.6) 100 (67.6) 17 (60.7) 0.481 Insulin dose (U/Kg) 0.59±0.20 0.60±0.22 0.55±0.16 0.269 Lipid-lowering drugs^c 81 (53.6) 68 (55.7) 9 (40.9) 0.199 ACEi or ARB use^d 112 (68.3) 94 (70.1) 14 (58.3) 0.252 Use of 3 or more

antihypertensive drugs^d 72 (43.9) 62 (46.6) 8 (33.3) 0.228

Data are shown as mean ± standard deviation, median (interquartile range) or number (percentage).

aMissing value n=42

b Missing value n=4

BMI, body mass index; BP; blood pressure; ACEi, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker.

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Table S3. Participants’ characteristics according to normalization of vibration perception thresholds at 6 months.

Data are shown as mean ± standard deviation, median (interquartile range) or number (percentage). Only participants with abnormal VPTs at baseline and with information at 6 months were included (n=86).

aMissing value n=16

b Missing value n=3

d Missing value n=13

Normalization of VPTs at 6 months p-value No

(n=56)

Yes (n=30)

Baseline clinical characteristics

Age at transplant (years) 42.4±6.5 38.8±5.8 0.013

Gender (Male) 45 (80.4) 23 (76.7) 0.688

BMI (Kg/m2) 23.8±3.1 23.7±2.8 0.838

<25 38 (67.9) 22 (73.3) 0.730

25-29.9 17 (30.4) 7 (23.3)

³30 1 (1.8) 1 (3.3)

Active smokers^a 12 (26.1) 6 (25.0) 0.921

Systolic BP (mmHg) 142±21 143±18 0.804

Diabetic retinopathy 55 (98.2) 30 (100) 0.462

Diabetes duration (years) 27.6±6.3 27.6±7.1 0.996

Patients receiving dialysis 39 (69.6) 23 (76.7) 0.489

VPTs (V) 29.6±7.3 24.4±4.8 0.001

Baseline laboratory characteristics

Total cholesterol (mg/dL) 184±57 167±48 0.188

Triglycerides (mg/dL) 109 (84-167) 88 (80-147) 0.096

LDL-cholesterol (mg/dl) 107±33 88±35 0.039

HbA1c (%) 7.7±1.7 7.2±1.1 0.178

HbA1c (mmol/mol) 61±19 55±12 0.178

Baseline treatment characteristics

Insulin dose (U/Kg) 0.63±0.22 0.59±0.24 0.522

ACEi or ARB use^d 31 (67.4) 16 (59.3) 0.484

Use of 3 or more

antihypertensive drugs^d 22 (47.8) 9 (34.6) 0.277

Transplantation-related variables

Induction immunosuppression Thymoglobulin

Basiliximab OKT3

19 (34.5) 32(58.2) 4 (7.3)

12 (40.0) 15 (50.0) 3 (10.0)

0.753

Maintenance immunosupression PDN+TAC+MMF

PDN+CsA+MMF 53 (94.6)

3 (5.4) 28 (93.3)

2 (6.7) 0.805

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ACEi, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker;

BMI, body mass index; BP; blood pressure; MMF, mycophenolate mofetil; PDN, prednisone; SRL, sirolimus; TAC, tacrolimus; VPTs, vibration perception thresholds.

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Table S4. Participants’ characteristics according to normalization of vibration perception thresholds at 2-3 years.

Data are shown as mean ± standard deviation, median (interquartile range) or number (percentage). Only participants with abnormal VPTs at baseline and with information at 2-3 years were included (n=68).

aMissing value n=17

b Missing value n=2

d Missing value n=12

Normalization VPTs at 2-3 years p-value No

(n=34)

Yes (n=34)

Baseline clinical characteristics

Age at transplant (years) 41.2±6.9 38.9±5.3 0.137

Gender (Male) 27 (79.4) 25 (73.5) 0.567

BMI (Kg/m2) 23.1±2.7 23.5±2.8 0.546

<25 28 (82.4) 25 (73.5) 0.483

25-29.9 6 (17.6) 8 (23.5)

³30 0 (0) 1 (2.9)

Active smokers^a 8 (29.6) 8 (33.3) 0.776

Systolic BP (mmHg) 143±20 142±19 0.712

Diabetic retinopathy 34 (100) 34 (100) 1.000

Diabetes duration (years) 25.9±6.9 27.1±6.0 0.434

Patients receiving dialysis 24 (70.6) 23 (67.6) 0.793

VPTs (V) 32.0±8.6 25.1±4.1 <0.001

Baseline laboratory characteristics

Total cholesterol (mg/dL) 198±66 176±32 0.133

Triglycerides (mg/dL) 128 (84-181) 112 (88-156) 0.474

LDL-cholesterol (mg/dl) 114±37 104±26 0.253

HbA1c (%) 7.9±1.9 7.6±1.3 0.421

HbA1c (mmol/mol) 63±21 59±14 0.421

Baseline treatment characteristics

Insulin dose (U/Kg) 0.60±0.21 0.66±0.25 0.272

ACEi or ARB use^d 17 (65.4) 20 (64.5) 0.945

Use of 3 or more

antihypertensive drugs^d 14 (53.8) 11 (36.7) 0.197

Transplantation-related variables

Induction immunosuppression Thymoglobulin

Basiliximab OKT3

14 (41.2) 17 (50)

3 (8.8)

11 (34.4) 17 (53.1) 4 (12.5)

0.801

Maintenance immunosupression PDN+TAC+MMF

PDN+CsA+MMF 31 (91.2)

3 (8.8) 32 (94.1)

2 (5.9) 0.642

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ACEi, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker;

BMI, body mass index; BP; blood pressure; MMF, mycophenolate mofetil; PDN, prednisone; SRL, sirolimus; TAC, tacrolimus; VPTs, vibration perception thresholds.

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Figure S1. Prevalence of orthostatic hypotension during follow-up.

p-values between several points of follow-up are shown.

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Figure S2. VPTs trajectories during 10 years of follow-up according to transplantation modality (SPK vs. PAK).

A: Mean values pretransplant, at 6 months, and 2-3, 5-6, and 8-10 years posttransplantation in SPK participants.

B: Mean values pretransplant, at 6 months, and 2-3, 5-6, and 8-10 years posttransplantation in PAK participants.

Mean with 95% confidence interval are shown.

p-values between several points of follow-up are shown.

PAK, pancreas after kidney; SPK, simultaneous pancreas-kidney; V, volts; VPTs, vibration perception thresholds.

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Figure S3. Pancreas graft survival according to VPTs status only in SPK recipients.

Cox-regression analysis adjusted for age, sex, diabetes duration at transplantation, previous cardiovascular disease, pretransplant body mass index and systolic blood pressure. HR for abnormal VPTs, 2.19 (1.06-4.54); p=0.035.

VPTs, vibration perception thresholds.

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Figure S4. Pancreas graft survival according to VPTs status at 6 months (A) and 2-3 years (B) only in SPK recipients.

Cox-regression analysis adjusted for age, sex, diabetes duration before

transplantation, previous cardiovascular disease, pretransplant body mass index, and systolic blood pressure.

A: HR for baseline abnormal – 6 months normal, 1.20 (0.36-4.01); p=0.765. HR for abnormal VPTs at 6 months, 3.30 (1.41-7.74); p=0.006.

B: HR for baseline abnormal – 2-3 years normal, 1.96 (0.72-4.88); p=0.196. HR for abnormal VPTs at 2-3 years, 2.11 (0.83-5.38); p=0.116.

VPTs, vibration perception thresholds.

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Figure S5: CVD events during the follow-up according to baseline diabetic neuropathy.

In those without baseline DN, peripheral vascular events (n=3) consisted of n=2 minor amputations (toe) and n=1 major amputation.

In those with baseline DN, peripheral vascular events (n=13) consisted of n=6 minor amputations, n=6 major amputations and n=1 revascularization.

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Figure S6. CVD-free survival according to pretransplant VPTs status only in SPK recipients.

Cox-regression analysis adjusted for age, sex, diabetes duration at transplantation, previous cardiovascular disease, pretransplant body mass index and systolic blood pressure. HR for abnormal VPTs, 2.57 [1.17-5.64]; p=0.018.

CVD, cardiovascular disease; VPTs, vibration perception thresholds.

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Figure S7. CVD-free survival according to VPTs status at 6 months only in SPK recipients.

Cox-regression analysis adjusted for age, sex, diabetes duration before

transplantation, previous cardiovascular disease, pre-transplant body mass index, type of transplant (simultaneous pancreas-kidney or pancreas after kidney) and systolic blood pressure. HR for baseline abnormal – 6 months normal, 0.83 (0.21- 3.26); p=0.784. HR for abnormal VPTs at 6 months, 3.16 (1.26-7.90); p=0.014.

CVD, cardiovascular disease; VPTs, vibration perception thresholds.