**1. Introduction**

Kidney transplantation is the best available treatment for patients with end-stage kidney disease [1,2]. In recent times, short-term graft survival has seen grea<sup>t</sup> improvement, which unfortunately has not been paralleled by equivalent improvement in long-term graft survival [3]. An important threat to long-term graft survival is progressive loss of kidney allograft function related to progressive fibrosis [4]. Despite its clinical importance, early identification of fibrosis appearance remains a challenge [5]. Currently, biopsy samples are the gold standard for the detection of established kidney fibrosis, but this has the evident drawback as a follow-up measurement of requiring an invasive procedure, which generates discomfort for the patients and can be complicated by bleeding. Other drawbacks are sampling variability and sampling errors [5,6]. Therefore, grea<sup>t</sup> interest exists in finding non-invasive biomarkers that can detect fibrosis formation, ideally at early stages [4].

Kidney allograft fibrosis reflects a pathological response to injury where the equilibrium between extracellular matrix formation and degradation is deregulated and progressive deposition of collagens, among other matrix constituents, takes place [7,8]. Assessment of active collagen formation may identify kidney transplant recipients (KTRs) at high risk of fibrosis progression and therefore development of chronic graft failure [9,10]. Among the different collagens, collagen type VI (COL VI) is found in the kidney and is constantly produced by fibroblasts at relative low levels in the interstitium, the intima and adventitia layers of the kidney vasculature, as well as in the glomeruli [11–13]. Under normal conditions, COL VI has an important physiological role in maintaining extracellular matrix (ECM) structure and function, controlling matrix and cell orientation [14]. However, under pathological conditions (e.g., chronic kidney disease), its active deposition in the kidneys is massively increased [9,12]. During production of COL VI, the C5 domain at the C-terminal of the α3 chain is released from the immediate pericellular matrix [15]. The PRO-C6 assay detects the C-terminal end of this domain and is proposed as a surrogate biomarker for COL VI active formation [9]. Moreover, the cleavage of part of this domain gives rise to a bioactive molecule, named endotrophin, which is also detected by the PRO-C6 assay [15,16]. Endotrophin has important biological effects, such as attracting macrophages, increasing transforming growth factor-β (TGFβ) signaling, promoting epithelial–mesenchymal transition, adipose tissue fibrosis, and metabolic dysfunction [17]. Increased plasma levels of PRO-C6 have previously been associated with the progression of chronic kidney disease and, specifically in the post-transplantation setting, with reduced graft function in KTR [4,9,18,19]. Whether associations between PRO-C6 and decreased graft function indeed correspond to increased fibrotic or inflammatory changes in the kidneys and whether it could be used as a non-invasive biomarker for fibrosis development in KTR remain unknown.

In the current study, we aimed to investigate the cross-sectional and longitudinal associations between PRO-C6 in plasma and urine, and proven histological changes in KTR of the minimization of maintenance immunosuppression early after kidney transplantation (MECANO) trial, which is a randomized, controlled, open-label, multicenter trial testing early cyclosporine A (CsA) elimination. Furthermore, since it is known that CsA nephrotoxicity includes pathological increased production and decreased degradation of extracellular matrix proteins, including collagen, and TGF-β up-regulation [20–22], we explored a potential differential role of PRO-C6 as a biomarker of fibrosis among patients under different immunosuppressive regimens.

#### **2. Materials and Methods**

#### *2.1. Study Design and Population*

Between November 2005 and June 2009, 361 de novo KTRs were recruited in three Dutch transplantation centers to participate in the MECANO trial (trial registration: NTR1615). The study was conducted according to the Good Clinical Practice guidelines, in accordance with the ethical principles of the Declaration of Helsinki, and was approved by the Dutch Medical Ethical Board for medical research (METC 04/154, 1 October 2004) [23,24]. All patients signed written informed consent forms. This study was a 24-month, prospective, multi-center, open-label, randomized, controlled trial, aiming at optimizing maintenance immunosuppression and reducing side e ffects. During the first six months after enrollment, all patients had a similar quadruple immunosuppressive regimen: induction with basiliximab, followed by CsA, mycophenolate sodium (MPS), and prednisolone [24]. At month six, a protocol biopsy was performed. When no histological signs of rejection were seen, patients were randomized to receive dual immunosuppressive therapy with CsA (*n* = 89), MPS, or everolimus (EVL) (*n* = 96), all in combination with prednisolone. In case of (borderline) rejection patients, were not randomized. The primary endpoint of the MECANO study was the development of interstitial fibrosis at the 24-months protocol biopsy.

After enrollment of 39 patients, the MPS-group was prematurely stopped by the Data Safety Monitoring Board because of an unacceptably high rejection percentage (21%). The trial continued as a two-group trial, comparing CsA and EVL. The results of the primary outcome of the study were published in 2016 [23].

#### *2.2. Protocol Kidney Biopsies and Histological Analyses*

Protocol biopsies were scheduled at 6 and 24 months after transplantation. At six months, biopsies were obtained in 99% and 98% of patients in the CsA group and the EVL group, respectively. Of the available biopsies, 78% and 81% in the CsA group and the EVL group were considered adequate, respectively. At 24 months, biopsies were obtained in 84% and 79% of patients in the CsA group and the EVL group, respectively. The prevalence of adequate samples was 81% and 73% in the CsA group and the EVL group, respectively (*p* = 0.4, two-tailed). The current study reports the results of the 94 patients (51 in the CsA group and 43 in the EVL group) whose 6-month biopsies met the minimal adequacy threshold of seven glomeruli and one artery.

Tissues were formalin-fixed and para ffin-embedded and stained with periodic-acid Schi ff diastase, hematoxylin/eosin, and Jones' methenamine silver. Two independent kidney pathologists (Amsterdam University Medical Center (UMC) and Leiden UMC, The Netherlands), unaware of any clinical data, classified the biopsies according to the 2015 update of the Ban ff classification [25] and assigned a Ban ff interstitial fibrosis/tubular atrophy (IF/TA) score. Morphometric analysis of cortical interstitial fibrosis was centralized at the Amsterdam UMC. Adequate protocol biopsy sections were stained with Picro Sirius Red (PSR, Aldrich, Munich, Germany), which is used for the detection of collagen fibers. PSR-stained slides were digitalized using a slide virtual microscope system (Olympus, Tokyo, Japan) with a 20× magnification objective and saved in Tagged Image File Format (TIFF format). Image analyses were performed with the ImageJ software package (National Institutes of Health, Bethesda, MD, USA) where the PSR-stained area was aut omatically assessed by means of a macro. All input was verified manually. Inflammation was evaluated by the total percentage of inflamed cortical area (ti-score) as a continuous score [26].

#### *2.3. PRO-C6 Detection*

Plasma and urine PRO-C6 concentrations were measured using a competitive enzyme-linked immunosorbent assay (Nordic Bioscience, Herlev, Denmark) that specifically detects the last 10 amino acids of the alpha-3 chain of COL VI (3168KPGVISVMGT3177) and is validated for both sample matrices [27]. The assay has a detection limit of 0.15 ng/mL and a 95% confidence interval for interand intra-assay variability in plasma samples reported as 3.4%–12.4% and 1.1%–5.3%, respectively [19]. For urine samples, the detection limit was the same as plasma, and inter- and intra-assay variability are reported as 7.9% and 3.2%, respectively [9]. To account for variations in urine concentration, urinary PRO-C6 was divided by urinary creatinine, measured by the QuantiChrom™ Creatinine Assay Kit (BioAssay Systems, Hayward, CA, USA), and the PRO-C6/creatinine ratio was used in all analyses.

## *2.4. Statistical Analyses*

Data analyses, computations, and graphs were performed with SPSS 25.0 software (IBM Corporation, Chicago, IL, USA). To test whether variables were normally distributed, a histogram was generated for each variable. For descriptive statistics data were presented as mean (standard deviation (SD)) for normally distributed data, and as median (interquartile range (IQR)) for variables with a non-normal distribution. Categorical data were expressed as number (percentage).

Differences in plasma and urine PRO-C6 and biopsy changes (IF/TA score, PSR, and ti-score) among subgroups of KTRs according to their treatment regimen and to their primary kidney disease were tested by one-way ANOVA for continuous variables with normal distribution, Mann–Whitney U test for continuous variables with skewed distribution, and X<sup>2</sup> test for categorical variables. Linear regression analyses were performed to study the association of plasma and urine PRO-C6 with biopsy changes at 6 and 24 months and the delta between the two visits. Furthermore, subgroup analyses were performed by dividing patients by the immunosuppressive regimen used. We also performed sensitivity analyses, in which patients who were grouped under "unknown cause" as primary kidney disease were recoded as if they have been suffering from glomerulonephritis as primary kidney disease. For all statistical analyses, a 2-sided *p* < 0.05 was considered significant.
