Hernández et al. BMC Nephrology (2022) 23:357 https://doi.org/10.1186/s12882-022-02989-z STUDY PROTOCOL Open Access Treatment of early borderline lesions in low immunological risk kidney transplant patients: a Spanish multicenter, randomized, controlled parallel-group study protocol: the TRAINING study Domingo Hernández1,2*†, Teresa Vázquez‑Sánchez1†, Eugenia Sola1, Veronica Lopez1, Pedro Ruiz‑Esteban1, Abelardo Caballero3, Eduardo Salido4, Myriam Leon5, Aurelio Rodriguez6, Nuria Serra7, Consuelo Rodriguez6, Carme Facundo7, Manel Perello8, Irene Silva7, Domingo Marrero‑Miranda6, Ignacio Cidraque8, Francesc Moreso8, Luis Guirado7, Daniel Serón8† and Armando Torres6† Abstract Background: Subclinical inflammation, including borderline lesions (BL), is very common (30–40%) after kidney transplantation (KT), even in low immunological risk patients, and can lead to interstitial fibrosis/tubular atrophy (IFTA) and worsening of renal function with graft loss. Few controlled studies have analyzed the therapeutic benefit of treat‑ ing these BL on renal function and graft histology. Furthermore, these studies have only used bolus steroids, which may be insufficient to slow the progression of these lesions. Klotho, a transmembrane protein produced mainly in the kidney with antifibrotic properties, plays a crucial role in the senescence-inflammation binomial of kidney tissue. Sys‑ temic and local inflammation decrease renal tissue expression and soluble levels of α-klotho. It is therefore important to determine whether treatment of BL prevents a decrease in α-klotho levels, progression of IFTA, and loss of kidney function. Methods: The TRAINING study will randomize 80 patients with low immunological risk who will receive their first KT. antithymocyte globulin (Grafalon®) (6 mg/kg/day) prevents or decreases the progression of IFTA and the worsening The aim of the study is to determine whether the treatment of early BL (3rd month post-KT) with polyclonal rabbit of BL with Grafalon® can modify the expression and levels of klotho, as well as the pro-inflammatory cytokines that of graft function compared to conventional therapy after two years post-KT, as well as to analyze whether treatment regulate its expression. † Daniel Serón and Armando Torres are share senior authorship. *Correspondence: domingohernandez@gmail.com 1 Nephrology Department, Hospital Regional Universitario de Málaga and University of Málaga, IBIMA, REDinREN (RD16/0009/0006 and RICORS RD21/0005/0012), E‑29010 Málaga, Spain Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hernández et al. BMC Nephrology (2022) 23:357 Page 2 of 10 and safety of Grafalon® treatment in low-immunological-risk KT patients with early BL. Discussion: This phase IV investigator-driven, randomized, placebo-controlled clinical trial will examine the efficacy Trial registration: clini​caltr​ials.​gov: NCT04936282. Registered June 23, 2021, https://​clini​caltr​ials.​gov/​ct2/​show/​ NCT04​936282?​term=​NCT04​93628​2&​draw=​2&​rank=1. Protocol Version 2 of 21 January 2022. Sponsor: Canary Isles Institute for Health Research Foundation, Canary Isles (FIISC). mgomez@​fciisc.​org. Keywords: kidney transplant, borderline lesions, α-klotho, low immunological risk, subclinical inflammation Background tubulo-interstitial lymphocytic infiltrate with few infec- Short-term outcomes after kidney transplantation (KT) tious complications. This should prevent long-term wors- have improved significantly, but 30% of grafts lose their ening of kidney graft function. function within 10 years of transplantation [1]. The pre- Finally, Klotho is a transmembrane protein pro- dominant histological lesion is a combination of inflam- duced mainly in the kidney that has antifibrotic and mation and interstitial fibrosis/tubular atrophy (IFTA) antiapoptotic properties, playing a crucial role in the [2–5]. Subclinical inflammation, including borderline aging-inflammation binomial of kidney tissue and pro- lesions (BL), is very common after KT (30–40%), even gression of chronic kidney disease (CKD) [21, 22]. In in patients with low immunological risk and stable graft fact, α-klotho deficiency in animal models and in human function. These lesions can lead to IFTA, worsening of studies is associated with early senescence, kidney fibro- graft function and loss of the KT, especially when inter- sis and accelerated arteriosclerosis [23, 24]. Systemic and stitial inflammation is associated with tubulitis [6–10]. local inflammation decrease renal tissue expression and However, there is lack evidence of treating BL lesions and soluble levels of α-klotho [25]. Likewise, proinflammatory slowering allograft function. A recent randomized study cytokines (TNF-α or TWEAK) induce less expression of by our group (Clini​calTr​ials.​gov, number NCT02284464), α-klotho, contributing to tissue damage [26]. Therefore, performed in recipients with a low immunological risk it is plausible that a sustained subclinical inflammation of with and without steroids, showed that those who had the grafts could contribute to the reduction in α-klotho BL at the third month post-KT presented more IFTA and and premature graft senescence. worse renal function at 24 months post-KT compared to It remains to be clarified whether anti-rejection treat- patients without inflammation, which was more preva- ment of BL, detected early in the third month post- lent in those who did not receive steroids [11]. KT, could prevent the decrease in α-klotho levels, the There is evidence that not treating these lesions can progression of IFTA and the loss of kidney function. lead to chronic histological changes and impaired graft Accordingly, it is reasonable to undertake a randomized function [10, 12], but most previous studies were not controlled clinical trial to assess the evolution of early BL Grafalon®. Determining a favorable therapeutic response controlled. Additionally, they did not focus on treatment and levels of α-klotho after anti-rejection therapy with for subclinical inflammation that did not reach the score for acute rejection ≥IA (i2,t2) of the Banff ’19 classifica- would provide valuable clinical information to improve tion [13]. Moreover, they mostly only used bolus ster- the long-term survival of KT in patients with low immu- oids, which could be insufficient to slow the progression nological risk. of these lesions [14, 15]. Therefore, the clinical impact of adequate anti-rejection treatment remains controver- Methods/Desing sial [7, 16]. Controlled studies are needed to assess the Objectives therapeutic response in patients with BL to improve graft Primary objectives Polyclonal rabbit antithymocyte globulins (Grafalon® survival. month post-KT, with Grafalon® prevents or reduces the Determine whether treatment of BL, detected at the third and Thymoglobulin®) are very effective for the preven- progression of IFTA and the deterioration of graft func- KT [17, 18]. Grafalon® (Neovii Pharmaceuticals AG) has tion and treatment of immunological dysfunction post- tion compared to conventional clinical follow-up, after a higher clearance compared to Thymoglobulin® (Sanofi two years post-KT. Genzyme) and is associated with a faster reconstitu- Secondary objectives tion of the lymphocytic subpopulations (CD3, CD4 and globulin® [19, 20]. Grafalon® could represent an effec- CD8) and generation of regulatory T cells than Thymo- 1. Analyze the incidence of immunologic dysfunction (acute clinical and subclinical rejections) and the his- tive therapeutic tool to suppress or minimize the early tological evolution. Hernández et al. BMC Nephrology (2022) 23:357 Page 3 of 10 2. Determine whether anti-rejection therapy of BL can Exclusion criteria modify klotho expression and levels and the pro- inflammatory cytokines that regulate its expression. a) Recipients of a multi-organ transplant. 3. Assess the association between α-klotho, urinary b) Re-transplants chemokine levels (CXCL9 and CXCL10), and acute c) Patients with biopsy-proven acute rejection or no clinical and subclinical rejection, as well as with the histological inflammation. IFTA lesions. d) Cold ischemia time > 30 hours. 4. Detect the generation of donor-specific anti-HLA e) Serum creatinine >2 mg/dl or proteinuria >1 g/day at antibodies (DSA). randomization. 5. Determine the impact of anti-rejection treatment on f ) Presence of significant thrombopenia (<100,000/ proteinuria during follow-up. mm3) or leukopenia (<3000 mm/3) at randomization. 6. Record graft and patient survival at the first and sec- g) Previous episode of clinical or subclinical rejection ond year of follow-up. (≥IA) before randomization. 7. Assess the adherence to immunosuppressive therapy h) Presence of BL before randomization and its impact on IFTA lesions and kidney function. i) CMV infection or disease in the first three months after transplantation j) BK-polyomavirus nephropathy at randomization. k) Recurrent or de novo glomerulonephritis. Trial Design l) Treatment with immunosuppressive drugs other This is a Spanish multicenter, investigator-driven, ran- than those in this clinical trial. domized, open-label, two-arm, clinical trial with a m) Patients who are positive for the human immunode- 24-month follow-up in patients with a first deceased ficiency virus or with severe systemic infection, who, donor or living donor KT who have BL at the time of the in the opinion of the investigator, require continued third-month protocol biopsy. The immunosuppressants therapy. in the study arms are those routinely used in clinical n) Previous (within the last 5 years) or present malignancy, practice. This is a phase IV clinical trial under the usual except excised basal or squamous cell carcinoma conditions (clini​caltr​ials.​gov: NCT04936282). Patient o) Pregnant or lactating women. recruitment time will be 12 months. The minimum fol- low-up period for patients will be-come 2 years, when a second protocol biopsy will be performed. Subject withdrawal criteria If the study is interrupted for any reason, the patient Eligibility Criteria and Exclusions will be considered to have discontinued treatment pre- Inclusion criteria maturely and will only be included in the “intention to treat” (ITT) analysis and will be eliminated from the a) Patients of either sex, older than 18 years, with low “per protocol” (PP) analysis. immunological risk (PRA < 20% and absence of DSA), All subjects who are excluded from the study for any who receive their first deceased donor or living donor reason will continue with the same follow-up as those KT. who remain in the protocol to perform a more robust b) Presence of BL, excluding isolated inflammation (t0, analysis by intention to treat. i > 0) and isolated tubulitis (t > 0, i0) c) Patients receiving tacrolimus in combination with mycophenolic acid (MPA) and steroids. Study Procedures and Work Plan d) Absence of clinical or subclinical and histological This study will be carried out in four Spanish transplant inflammation-related allograft dysfunction before centers (Hospital Regional Universitario de Málaga; randomization Hospital Universitario de Canarias, Tenerife; Hospital e) Absence of de novo DSA anti-HLA antibodies at the Vall d’Hebron, and Hospital Fundacio Puigvert, Barce- time of randomization. lona) within the Kidney Research Network (RICORS). f ) Provision of written informed consent (obtained by Any modification to the protocol will be approved by the attending physician). the relevant Ethics and Research Committee and the g) Acceptance of efficient contraception in women. Spanish Drug Agency and notified as necessary. Hernández et al. BMC Nephrology (2022) 23:357 Page 4 of 10 Randomization • Anti-CMV (except in the case of negative IgG Subjects will be randomized to one of the arms in a recipient and donor) and Pneumocystis prophy- 1:1 ratio, carried out locally in each center through the laxis must be given. electronic data collection notebook (e-DCN) that will include a computer program for the generation of ran- DSA will be monitored (every 3 months in the first dom numbers. If a patient is randomized but does not year and every 6 months in the second year). Addition- receive the study treatment, they will not be included ally, immunosuppression will be maintained throughout in either the ITT or the PP analysis. Their number and the study (post-randomization) following the therapeutic treatment group will not be reused for new patients. schedule: Treatment Arms • Steroids 5 mg/day Study Arms: control (Group I) • Maintain levels of tacrolimus (8–10 ng/ml) to achieve a proper immunosuppression during study. • Tacrolimus: Advagraf® 0.15 mg/kg/day or Envar- • MMF doses of at least 1 g/day or MPA 720 mg/day sus® (0.1 mg/kg/day) to maintain levels of 8–12 ng/ • Do not administer anti-mTOR drugs ml at the first month. Thereafter Advagraf® 0.1 mg/ kg/day or Envarsus® 0.07 mg/kg/day to maintain In both therapeutic arms, induction treatment with levels of 6–8 ng/ml. anti-CD25 monoclonal antibodies (Basiliximab 20 mg i.v., • Mycophenolate mofetil (MMF) 2 g/day or days 0 and 4) or polyclonal antibodies (Thymoglobulin mycophenolic acid (MPA) 1440 mg/day the first 1 mg/kg/day for 4–7 days) will be allowed in patients at 30 days post-KT. Afterwards MMF 1 g/day or MPA risk of delayed graft function. 720 mg/day. Patients without inflammation (i0,t0) at the third • Steroids: 0.5 g Methylprednisolone pre-KT and month protocol biopsy, or with just isolated inflammation 125 mg on day 1; prednisone 30 mg the first 15 days (i > 0,t0) or isolated tubulitis (t > 0,i0), will continue con- post-KT and then 5 mg/week until reaching 5 mg/ ventional immunosuppression (steroids+Tac + MMF) day at the 2nd month post-KT and the same dose and be monitored until study end, performing the proto- will be continued. col biopsy at month 24, in order to determine the natural history of the histological changes and the evolution of This treatment will continue until randomization. renal function, comparing them to patients with BL and randomized to the two therapeutic arms. Immunosuppressants will be prescribed and adminis- Study Arms: Intervention (Group II) tered according to routine clinical practice, so their pack- I, Group II will receive treatment with Grafalon® as Apart from the standard treatment taken in Group aging and labeling is approved for marketing. - Grafalon® 6 mg/kg/day in a single day. Its adminis- follows. Other treatments tration will be done as follows: Acute rejection will be treated with 3 boluses of 500 mg/ day of methylprednisolone i.v. Corticosteroid resistant rejections will be treated with a standard 7–10 day cycle Solumoderin® 125 mg i.v. and antihistamine H1 • Pre-medication before the first and second doses: of thymoglobulin. Acute humoral rejections will be antagonist (dexchlorpheniramine [Polaramine® 1 treated according to the protocol of each center, whether vial (5 mg) i.v.], diphenhydramine, or other from it is plasmapheresis, immunoglobulins, and rituximab, alone or in combination. • Grafalon®: Dilute the calculated dose in 500 ml of the same therapeutic group). Drugs to prevent the most frequent infections such as physiological serum and administer within 8 hours Pneumocystis jirovecii and CMV will be used accord- through a central line or A-V fistula. ing to the protocol of each center. Dyslipidemia will be • Administer half the dose if leukocytes <3000/mm3 treated with statins, or with ezetimibe when adverse and platelets <80,000/mm3. Stop when leukocytes effects appear. Fibrates should be avoided, with the exception of gemfibrozil, as they can be associated with • During treatment with Grafalon®: <1500/mm3 or platelets <40,000/mm3. impaired kidney function. Hypertension will be treated according to the protocol of each center to obtain a target • MMF/MPA: 500 mg/12 h or 360 mg/12 h, moni- BP <130/80 mmHg. The treatment of post-transplant dia- toring leukocyte and platelet counts. betes will be carried out according to the usual therapeu- tic indications of this entity. Hernández et al. BMC Nephrology (2022) 23:357 Page 5 of 10 Safety committee and Safety monitoring Biopsies corresponding to the pre-randomization and This is composed of those responsible for each center. final visits will also be sent to the central pathologist at Periodically and via teleconference, adverse effects will be the Hospital Universitario de Canarias, who will give analyzed in both study groups. Interim safety analysis is a second interpretation based on the same Banff/19 preplanned by the sponsor during study, to the independ- criteria. ent data monitoring and safety committee. The score sum of the classification of chronic lesions In addition, the sponsor may end the study at any time will be obtained through 5 histological characteristics: and for any reason. a. cg: chronic glomerulopathy b. ci: chronic interstitial Definitions of end points c. ct: tubular atrophy (chronic) Primary end points: Presence of IFTA and graft function d. cv: arteriolar hyalinosis (chronic) at 24 months in both therapeutic arms. e. mm: mesangial matrix Main efficacy end points: renal function and histologi- cal lesions. Chronic injuries will be defined as IFTA scores ≥2 (ci + ct) according to the Banff ’19 classification. The score a) The glomerular filtration rate (GFR) determined by sum of all chronicity compartments (cg + ci + ct + cv) CKD-EPI formula. will also be taken into account. b) Appearance of acute or chronic pathological lesions Secondary efficacy variables: on allograft biopsy. Biopsies will be performed at the a) Graft and patient survival. third month post-transplantation and at month 24 b) Biopsy-proven acute rejection rate, and proteinuria post-randomization. They will be evaluated accord- at 3,6,12 and 24 months. ing to the Banff ’19 classification [13]. For a correct c) Blood pressure (BP) and number of hypotensive interpretation of the biopsies, at least 10 glomeruli drugs, lipid levels and need for lipid-lowering drugs, and 2 artery will be required. weight gain and body mass index. d) Post-transplant diabetes rate or glucose intolerance The different histological diagnostic categories will be: according to ADA criteria [28]. e) Adherence to immunosuppressants: evaluated using 1. No inflammation (i0,t0) the BAASIS scale at each visit of the study by interview 2. BL, defined as: [29]. -Serum levels of α-klotho will be measured by ELISA using a specific kit for soluble human α-klotho (Immuno- • Foci of tubulitis (t1,t2,t3) with mild inflammation Biological Lab.). This assay has an inter- and intra-assay (i1), or mild tubulitis (t1) with moderate-to-severe coefficient of variability of 5% and 5.0–7.3%, respectively, inflammation (i2 or i3), but below the threshold for and a lower limit of detection of 3.0 relative units/ml. Banff IA rejection (i2,t2). Therefore, the possible Klotho will also be measured in urine [25]. Circulating combinations of BL scores are: i1t1, i2t1, i3t1, i1t2 FGF-23 levels will be measured using an ELISA kit for and i1t3. human (C-terminal) FGF-23 (Immunotopics). Plasma • Absence of transmural arteritis (v0) levels of soluble TNF-alpha and TWEAK/Fn14 will also be determined by ELISA with a specific kit [30, 31]. determined with an immunoassay kit (Milliprex®). The Due to the excellent prognosis of lesions with isolated -Urinary chemokines CXCL9 and CXCL10 will be inflammation (i > 0, t0) [10] and isolated tubulitis (i0, t > 0) [27], patients with these lesions will not be included intra-assay coefficient of variation is <10%, and the inter- in the randomized arms of the study. assay <15%. A graft biopsy will be performed at any time during the study if graft dysfunction is detected, evidenced by pro- Methods and schedule for evaluating efficacy parameters teinuria ≥0.5 g/day or an increase in serum creatinine The study schedule is shown in Tables 1 and 2. Figure 1 ≥25% over baseline value. Biopsies for clinical indica- displays the scheme and determinations of the study. tion and those done pre-randomization will be assessed in each participating center. Local pathologist interpreta- tion will be performed to analyze the presence of clinical Safety assessment or subclinical rejection, BL or the progression of chronic All adverse events (AEs) will be monitored during lesions that suggest an eventual graft dysfunction. follow-up. Hernández et al. BMC Nephrology Table 1 Study visits and evaluation schedule Visit Baseline Month 1 Month 2 Month 3 Month 4 Month 6 Month 9 Month 12 Month 18 Month 24 KT Pre- Randomization randomization (2022) 23:357 Screening 1 2 3 4 5 6 7 8 9 10 Informed consent X Clinical and demographic data X X X X X X X X X X Pregnancy test X X Randomization criteria X X Clinical incidents (hypertension, diabetes, dyslipidemia) X X X X X X X X X X Kidney graft biopsy X X General lab testsa X X X X X X X X X X Anti-HLA (DSA) antibodies X X X Renal function X X X X X X X X X (CKD-EPI) Proteinuria X X X X X X X X X Urinary chemokines (CXCL9 and CXCL10) X X X X X Elisa Kits: Klotho, FGF-23 TNF and TWEAK/Fn14 X X X X X Assessment of therapeutic compliance (BAASIC scale) X X X a blood glucose, Calcium, creatinine, Phosphorus, Hematocrit, Hemoglobin, HDL-Cholesterol, LDL-Cholesterol, parathyroid hormone, tacrolimus levels, Total Cholesterol, triglycerides Page 6 of 10 Hernández et al. BMC Nephrology (2022) 23:357 Page 7 of 10 Table 2 Visiting schedule Activity Visits 0 1 2 3 4 5 6 7 8 9 10 Donor demographic data X Graft characteristics X Recipient clinical and demographic data X • Clinical and laboratory data: X X X X X X X X X • Anthropometric parameters • Delayed graft function • Anticalcineurin levels • Fasting blood glucose and hypoglycemic treatment • Serum creatinine • Estimated filtration (CKD-EPI) • Ultrasensitive C-reactive protein (PCR) • Acute rejection ○ Histological Type (Banff Classification ‘19) • Data ○ Treatment received ○ Corticosensitive/Corticoresistant ○ Serum creatinine at the start of treat- ment • BP, total cholesterol, HDL-cholesterol and LDL-cholesterol values • Proteinuria: protein / creatinine ratio or 24-hour proteinuria • Infections • Medical and surgical complications • Concomitant drugs Informed consent X Protocol biopsy X X Anti-HLA antibodies X X X X X X Assessment of therapeutic compliance X X X Fig. 1 Scheme and determinations of the study Hernández et al. BMC Nephrology (2022) 23:357 Page 8 of 10 The Safety Committee is composed of those responsi- Discussion ble at each participating center. A project safety report This study will provide information on the progres- will be drawn up every six months. The Safety Commit- sion of IFTA and the relationship between klotho lev- tee will analyze whether is it safe to continue with the els and subclinical inflammation (specifically BL) in study based on the cumulative number of cases. patients with a low immunological risk, evidenced at the The requirements of Spanish Royal Decree 1090/2015 3-month protocol biopsy. We will also assess other pro- (article 51) on the notification of adverse reactions will be inflammatory molecules and chemokines related with followed. In particular, any serious adverse event should the inflammation-senescence binomial, furthering our be reported to the electronic Case Report Form (eCRF) understanding of the pathogenic mechanisms that lead to within 24 hours and reported to all participating centers. accelerated graft senescence when subclinical inflamma- Required data for the analysis of AEs will be registered tion is present. This study can contribute to establishing by the investigator in an eCRF using a real-time data a new therapeutic approach in these patients, in order to recording system “on-line”. improve long-term post-KT outcomes. It will allow us to compare the natural history of the histological changes of Data monitoring, management, access and dissemination patients without inflammation with those with early BL Data monitoring will be undertaken by the Spanish Clini- included in the therapeutic arms. cal Research Network (SCReN) in collaboration with tion when not receiving Grafalon®. However, there is no Group I patients could have a higher risk of acute rejec- Health Institute Carlos III. Data will be recorded by each centre in the general database, and access will be con- consensus on the ideal treatment of BL detected in pro- trolled by Spanish law (Royal Decree 1090/2015). The tocol biopsies, nor are there sufficient controlled clinical results will be communicated through the usual channels. trials to conclusively demonstrate that not treating BL lesions can trigger acute immune dysfunction [7, 16]. In Data analysis and statistics any case, the risk of acute rejection and the generation of Sample size DSA can be minimized if we consider that only subjects Data analyzed in previous studies where renal function is with a low immunological risk will be included and will compared in patients with BL versus those without inflam- receive as induction anti-CD25 (Basiliximab) or poly- mation have been used as reference points [32, 33]. To detect clonal antibodies (Thymoglobulin) in case of a higher risk a difference in GFR of 10 ml/min at 24 months post-KT in of delayed graft function. In addition, anti-HLA antibod- both groups with an estimated standard deviation in one of ies will be closely monitored and a series of therapeutic the groups of 15 ml/min, a power of 80% (Beta = 0.20), and measures will be carried out aimed at minimizing immu- a statistical risk of 5% (alpha = 0.05), a total of 70 patients nological risk throughout the study. Moreover, we will with BL (35 in each therapeutic arm) is required. Assuming closely monitor immunosuppression-related side effects a 15% loss to follow-up, 80 patients (40 in each group) are such as diabetes and life-threatening complications (car- needed for the evaluation of efficacy (Epidat 3.1). diovascular disease or infections) [28]. This study will be carried out at four Spanish transplant Statistical analysis centers with intense transplant activity, which guarantees Descriptive statistics will be presented with the statisti- reaching the required sample size. In addition, Spain has cal analyses. An intention-to-treat and end-treatment the highest donor and transplant rate in the word. Thus, analysis will be performed. Categorical variables will be a sufficient number of KT with early BL is expected in analyzed according to the chi-square test or Fisher’s exact our study. Although the information will be taken from test. Quantitative data will be analyzed using the Student’s records of each hospital, which could lead to information t-test or the Mann-Whitney U test. In the case of three bias, the exhaustive follow-up and the collection of infor- quantitative samples, ANOVA or Kruskal-Wallis will be mation in a centralized electronic platform should mini- used as appropriate. Changes in the evolution over time mize this concern. of renal function in the two study groups will be analyzed In conclusion, this Spanish multicenter, investigator- using the general linear model for repeated measures, driven, randomized, placebo-controlled phase IV clini- adjusting for confounding variables. Graft and patient cal trial will assess the efficacy and safety of treatment of survival curves will be calculated by the Kaplan-Meier early BL with polyclonal rabbit antithymocyte globulin in method. The survival rates will be compared between low immunological risk KT patients. groups with the log-rank test method. A linear regression analysis will be done to determine the factors associated Abbreviations with changes in kidney function and the progression of AEs: Adverse events; BL: Borderline lesions; BP: Blood pressure; CKD: Chronic chronic lesions. Data will be analyzed using SPSS 20.0. kidney disease; DSA: Donor-specific anti-HLA antibodies; eCRF: Electronic Case Hernández et al. BMC Nephrology (2022) 23:357 Page 9 of 10 Report Form; GFR: Glomerular filtration rate; IFTA: Interstitial fibrosis/tubular 2. Riella LV, Djamali A, Pascual J. Chronic allograft injury: Mechanisms and atrophy; ITT: Intention to treat; KT: Kidney Transplantation; MMF: Mcopheno‑ potential treatment targets. Transplant Rev (Orlando). 2017;31(1):1–9. late mofetil; MPA: Mycophenolic acid; PP: Per protocol. 3. Kasiske BL, Gaston RS, Gourishankar S, Halloran PF, Matas AJ, Jeffery J, et al. Long-term deterioration of kidney allograft function. Am J Trans‑ Acknowledgments plant. 2005;5(6):1405–14. We are also grateful to the kidney transplant teams of the hospitals involved in 4. Nankivell BJ, Borrows RJ, Fung CL, O’Connell PJ, Allen RD, Chapman the study and thank Ian Johnstone for help with English language editing. JR. The natural history of chronic allograft nephropathy. N Engl J Med. 2003;349(24):2326–33. Authors’ contributions 5. García-Carro C, Dörje C, Åsberg A, Midtvedt K, Scott H, Reinholt FP, et al. D.H., T.V-S., E.S.,V.L., P.R-E., A.C., E.S., M.L., A.R., N.S., C.R., C.F., M.P., I.S., D.M-M., I.C., Inflammation in Early Kidney Allograft Surveillance Biopsies With and F.M., L.G., D.S., A.T. all contributed to the design and writing of this study proto‑ Without Associated Tubulointerstitial Chronic Damage as a Predictor col. All authors have read and approved the final manuscript. of Fibrosis Progression and Development of De Novo Donor Specific Antibodies. Transplantation. 2017;101(6):1410–5. Funding 6. Ortiz F, Gelpi R, Helanterä I, Melilli E, Honkanen E, Bestard O, et al. The study will receive grants from the Instituto de Salud Carlos III, Decreased Kidney Graft Survival in Low Immunological Risk Patients Madrid, Spain (IC121/00042 –general trial expenses; REDinREN Network, Showing Inflammation in Normal Protocol Biopsies. PLoS One. RD16/0009/0006, RD16/0009/0030 and RD16/0009/0031 and RICORS2040, 2016;11(8):e0159717. RD21/0005/0012 and RD21/0005/0016), FONDOS FEDER -all grants related to 7. Rush DN, Gibson IW. Subclinical Inflammation in Renal Transplantation. financing of trial personnel). Transplantation. 2019;103(6):e139–45. The funding bodies will play no role in the design of the study and collection, 8. Nankivell BJ, Agrawal N, Sharma A, Taverniti A, P’Ng CH, Shingde M, et al. analysis, and interpretation of data and in writing the manuscript. The clinical and pathological significance of borderline T cell-mediated rejection. Am J Transplant. 2019;19(5):1452–63. Availability of data and materials 9. Moreso F, Ibernon M, Gomà M, Carrera M, Fulladosa X, Hueso M, et al. Not applicable. Subclinical rejection associated with chronic allograft nephropathy in protocol biopsies as a risk factor for late graft loss. Am J Transplant. 2006;6(4):747–52. Declarations 10. Mehta RB, Tandukar S, Jorgensen D, Randhawa P, Sood P, Puttarajappa C, et al. Early subclinical tubulitis and interstitial inflammation in Ethics approval and consent to participate kidney transplantation have adverse clinical implications. Kidney Int. The study was conducted according to the guidelines of the Declaration of 2020;98(2):436–47. Helsinki, and approved by the Provincial Ethics and Clinical Research Com‑ 11. Hernández D, Alonso-Titos J, Vázquez T, León M, Caballero A, Cobo MA, mittee of Malaga (CEI Provincial Malaga) and by the Spanish Drug Agency et al. Clinical Relevance of Corticosteroid Withdrawal on Graft Histologi‑ (protocol code: TRAINING and date of approval: 08 March 2022). cal Lesions in Low-Immunological-Risk Kidney Transplant Patients. J Clin Informed consent will be obtained in writing from all subjects involved in the Med. 2021;10(9):2005. study. 12. Mehta R, Bhusal S, Randhawa P, Sood P, Cherukuri A, Wu C, et al. Short- term adverse effects of early subclinical allograft inflammation in kidney Consent for publication transplant recipients with a rapid steroid withdrawal protocol. Am J Not applicable. Transplant. 2018;18(7):1710–7. 13. Loupy A, Haas M, Roufosse C, Naesens M, Adam B, Afrouzian M, et al. Competing interests The Banff 2019 Kidney Meeting Report (I): Updates on and clarification The authors declare no conflict of interest. of criteria for T cell- and antibody-mediated rejection. Am J Transplant. 2020;20(9):2318–31. Author details 14. Beimler J, Zeier M. Borderline rejection after renal transplantation–to treat 1 Nephrology Department, Hospital Regional Universitario de Málaga or not to treat. Clin Transpl. 2009;23(Suppl 21):19–25. and University of Málaga, IBIMA, REDinREN (RD16/0009/0006 and RICORS 15. Kee TY, Chapman JR, O’Connell PJ, Fung CL, Allen RD, Kable K, et al. Treat‑ RD21/0005/0012), E‑29010 Málaga, Spain. 2 Nephrology Department, Carlos ment of subclinical rejection diagnosed by protocol biopsy of kidney Haya Regional University Hospital, Avda. Carlos Haya s/n., E‑29010 Malaga, transplants. Transplantation. 2006;82(1):36–42. Spain. 3 Immunology Department, Hospital Regional Universitario de Málaga 16. Park SH, Friedewald JJ. Are borderline changes real rejection? Current and University of Málaga, IBIMA, REDinREN (RD16/0009/0006 and RICORS viewpoints. Curr Opin Nephrol Hypertens. 2020;29(6):656–62. RD21/0005/0012), E‑29010 Malaga, Spain. 4 Pathology Department, Hospital 17. Opelz G, Naujokat C, Daniel V, Terness P, Döhler B. Disassociation between Universitario de Canarias, Instituto de Tecnologías Biomédicas-Universidad La risk of graft loss and risk of non-Hodgkin lymphoma with induction Laguna, E‑38320 Tenerife, Spain. 5 Pathology Department, Hospital Regional agents in renal transplant recipients. Transplantation. 2006;81(9):1227–33. Universitario de Malaga, IBIMA, REDinREN (RD16/0009/0006 and RICORS 18. Brennan DC, Daller JA, Lake KD, Cibrik D, Del Castillo D. Rabbit antithymo‑ RD21/0005/0012), E‑29010 Málaga, Spain. 6 Nephrology Department, Hospital cyte globulin versus basiliximab in renal transplantation. N Engl J Med. Universitario de Canarias, Instituto de Tecnologías Biomédicas-Universidad La 2006;355(19):1967–77. Laguna, REDinREN (RD16/0009/0031), E‑38320 Tenerife, Spain. 7 Nephrology 19. Oostenbrink LVE, Jol-van der Zijde CM, Kielsen K, Jansen-Hoogendijk Department, Fundación Puigvert, E‑08025 Barcelona, Spain. 8 Nephrology AM, Ifversen M, Müller KG, et al. Differential Elimination of Anti-Thymo‑ Department, Hospital Universitari Valld’Hebron and Universitat Autonoma, cyte Globulin of Fresenius and Genzyme Impacts T-Cell Reconstitu‑ REDinREN (RD16/0009/0030 and RICORS RD21/0005/0016), E‑08035 Barcelona, tion After Hematopoietic Stem Cell Transplantation. Front Immunol. Spain. 2019;10:315. 20. Shimony O, Nagler A, Gellman YN, Refaeli E, Rosenblum N, Eshkar-Sebban Received: 9 June 2022 Accepted: 24 October 2022 L, et al. Anti-T lymphocyte globulin (ATG) induces generation of regula‑ tory T cells, at least part of them express activated CD44. J Clin Immunol. 2012;32(1):173–88. 21. Izquierdo MC, Perez-Gomez MV, Sanchez-Niño MD, Sanz AB, Ruiz-Andres O, Poveda J, et al. Klotho, phosphate and inflammation/ageing in chronic kidney disease. 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