SUPPLEMENTAL DIGITAL CONTENT 1 (SDC 1)
Transplantation and Immunosuppression
Organs for transplantation were obtained from deceased donors through the “North Italia Transplant” organ procurement consortium (NITp, Milan). After induction with ATG (thymoglobulin, IMTIX, SANGSTAT), immunosuppression was maintained using cyclosporine (through levels between 100-250 ng/ml) or FK506 (through levels between 10-15 ng/ml), mycophenolate mofetil (500-2000 mg/day), and methylprednisolone (10 mg/day). Steroids were withdrawn within 3-6 months after transplantation.
Dyslipidemia and hypertension were treated with conventional treatments as suggested by KDOQI guidelines in T1D+ESRD (1) and KDIGO guidelines in KP and KD patients (2). In particular, dyslipidemia was efficiently controlled by statins and normal blood pressure was maintained with diuretics and/or ACE-inhibitors and/or angiotensin receptor blockers and/or calcium antagonists.
Hormone measurements
For the specific purpose of this study, in order to reflect common practice of a clinical biochemistry laboratory, we measured circulating hormones using commercially available analytical methods and the same laboratory was used for all samples. In this context, a single venous blood sample was drawn from each participant between 8 am and 10 am, in compliance with the Clinical and Laboratory Standards Institute (CLSI, previously National Committee for Clinical Laboratory Standards) guidelines (3), and kept at 4°C until serum was separated by centrifugation at 4°C. Serum aliquots were then stored at –80°C until assay.
On each sample we measured free-triiodothyronine (fT3), free-thyroxine (fT4), thyroid-stimulating hormone (TSH), prolactin, follicule-stimulating hormone (FSH), luteinizing hormone (LH), total (tT) and free testosterone (fT), ∆4-androstenedione (∆4A), dehydroepiandrosterone sulphate (DHEAS), sex hormone–binding globulin (SHBG), and 17β-estradiol (E2). Free-T3 and fT4 were measured by a competitive enzyme immunoassay (AIA-PACK fT4 and AIA-PACK fT3; TOSOH Corporation, Tokyo, Japan). TSH was measured by a two-site immunoenzymatic assay (AIA-PACK TSH 3rd-Gen; TOSOH Corporation, Tokyo, Japan). Prolactin, FSH, LH, and total T were measured by a direct chemiluminescence immunoassay (prolactin: ADVIA:Centaur; FSH: ADVIA:Centaur; LH: ADVIA:Centaur; testosterone: ADVIA:Centaur; Bayer Corporation, Tarrytown, NY, USA, today Siemens Medical Solutions Diagnostics). Commercial RIA kits were used to measure free T (125I Radioimmunoassay; Gamma Counter MDA 312 – Kontron, ADALTIS Italia) and 4A (125I Radioimmunoassay; DRG Androstenedione RIA-1539; ADALTIS Italia). An immunometric microplate assay was used for DHEAS (Enzyme Immunoassay; DiaSorin s.r.l., Saluggia, Italy). SHBG levels were measured using a solid-phase, chemiluminescent immunometric assay on Immulite 2000 (Medical Systems Corp., Genoa, Italy). Finally, E2 was measured by an heterogeneous competitive magnetic separation assay (Bayer Immuno 1 System, Bayer Corporation, Tarrytown, NY, USA, today Siemens Medical Solutions Diagnostics).
Venous blood samples for glycated haemoglobin (HBA1C) were also collected from all patients and controls on the day when they were invited to participate in the study. In hemodialyzed patients, blood samples were collected before dialytic treatment to avoid the confounding effect mediated by heparin administration and by the contact with hemodialysis membrane.
Quantitative penile sensory thresholds measurements
Penile skin thermal and vibratory sensitivity thresholds were comprehensively assessed by means of the Genito Sensory Analyzer (GSATM, Medoc Israel) (4), a dedicated tool that specifically delivers thermal and vibratory stimuli and simultaneously quantitatively measures peripheral sensory thresholds at different genital sites. Hand thermal and vibratory sensation thresholds were first tested at the pulp of the right index finger in both groups. Fingertip measurements are considered useful because they provide information about general neuropathy; likewise, they tend to relax the patient before he is tested on the penis (5). The measurements thus began at the genital level, including lateral aspects of the flaccid penile shaft (i.e., below the sulcus) and the glans penis with foreskin retracted, bilaterally. As previously suggested by Vardi et al. (4) stimuli were provided in an escalating manner until the man first reported a sensation by pressing a button. All the men were tested five times. For the specific purposes of the study, we used the mean±standard error (SE) of the five assessments, for both sides of the penile shaft.
Penile hemodynamic assessment
Dynamic penile color Doppler sonography, using intracavernosal injection of prostaglandin E1 (PGE1), plus audiovisual and manual genital stimulation, was also performed in all patients in order to assess penile hemodynamic. The test was conducted according to the protocol previously reported by Montorsi et al. (6), with the specific purpose to obtain an erection comparable to the maximal physiological erection (that is the erection obtained at home during sexual activity). In this context, cavernosal peak systolic velocity (PSV) and resistive index (RI) were bilaterally evaluated 20 min after intracavernosal PGE1 injection (6). For the specific purposes of the study, we used the mean (SE) for both sides of the penile shaft.
References
1)KDOQI guidelines. Am J Kidney Dis 2007;49 (Issue 2 SUPPL 2).
2)KDIGO guidelines. Am J Transplant 2009; 9 (Suppl 3).
3)National Committee for Clinical Laboratory Standards. How to define, determine, and utilize reference intervals in the clinical laboratory; approved guidelines. NCCLS document C28-A. Villanova (PA): NCCLS; pp59 June 1995.
4)Vardi Y, Gruenwald I, Sprecher E, Gertman I, Yartnitsky D. Normative values for female genital sensation. Urology 2000 Dec 20;56(6):1035-40.
5)Bemelmans BL, Hendrikx LBPM, Koldewijn EL, Lemmens WAJG, Debruyne FMJ, Meuleman EJH. Comparison of biothesiometry and neurophysiological investigations for the clinical evaluation of patients with erectile dysfunction. J Urol 1995 May;153(5):1483-6.
6)Montorsi F, Guazzoni G, Barbieri L, Ferini-Strambi L, Iannaccone S, Calori G, Nava L, Rigatti P, Pizzini G, Miani A. Genital plus audiovisual sexual stimulation following intracavernous vasoactiveinjection versus re-dosing for erectile dysfunction--results of a prospective study. J Urol 1998 Jan;159(1):113-5.