**4. Discussion**

This study shows, in a homogeneous well-characterized cohort of KTRs who were participants of the MECANO clinical trial, that 6-month post-transplant plasma concentration of PRO-C6 associates with graft biopsy-proven fibrotic and inflammatory changes, both cross-sectionally (IF/TA score) and longitudinally (IF/TA score and ti-score). Further, we show that these same associations are not found with 6-month urine PRO-C6, and that at 24 months, no cross-sectional association was present between fibrotic changes and either urine or plasma PRO-C6. Subgroup analyses comparing patients under CsA vs. EVL immunosuppressive therapy showed higher urinary concentration of PRO-C6 in the EVL group compared to the CsA groups during follow-up, despite lower fibrosis scorings.

The progression of kidney diseases is characterized by the appearance of progressive fibrosis, which reflects a pathological disequilibrium between the synthesis and degradation of ECM constituents, including collagens, within scarred kidneys [8,28]. COL VI is an ECM molecule distributed in the kidney interstitium, vasculature, and in the glomeruli, which is constantly produced by fibroblasts at relative low levels [12,13]. Under healthy conditions, it has an important physiological role in maintaining structure and function of the ECM by controlling organization and cell orientation [14]. However, its markedly increased synthesis and deposition has been reported under a wide spectrum of kidney pathologies [29,30].

COL VI biosynthesis and assembly involves a complex multi-step pathway [14,31]. During active deposition in the ECM, a pro-peptide in the α3 chain of COL VI is released; in turn, this gives rise to the bioactive molecule endotrophin [15,27], which is known to have a role in shaping a pro-inflammatory and pro-fibrotic microenvironment by, amongs<sup>t</sup> other processes, triggering an increase in cytokines such as TGFβ [16]. The PRO-C6 assay measures both the release of endotrophin and of the pro-peptide, reflecting newly formed molecules of mature COL VI [9,27]. In the post-transplantation setting, the assessment of active collagen formation has been proposed as a way of early identifying KTRs that are at high risk of fibrosis progression [9,10], and since allograft function loss is closely related to the appearance and progression of interstitial fibrosis and tubular atrophy [10,32,33], it could identify also KTRs at future risk of developing chronic graft failure [9,10].

Clinically, increased deposition of COL VI has been reported in multiple scenarios of chronic kidney disease [28,31], and specifically in the post-transplantation setting, a strong association was found between increased plasma concentration of PRO-C6 and a decrease in graft function over time [4]. In agreemen<sup>t</sup> with this evidence, we found a positive prospective association between 6-month PRO-C6 concentration and biopsy evidence of increased graft fibrosis (IF/TA). However, no associations were found with PSR staining. Following the evidence that patients receiving CsA are at risk of developing nephrotoxicity, which is also a condition with unregulated ECM deposition and TGF-β upregulation [20–22,34], we performed exploratory analysis by subgroups of immunosuppressive therapy. When dividing the population into subgroups, we found that patients in the CsA group had higher PSR% at 24 months, but urine PRO-C6 was higher in the EVL group.

This analysis shows that PRO-C6 measurement, as reflection of collagen VI synthesis, is associated with, but not identical to, quantification of fibrosis in transplanted kidneys, especially not under di fferent treatment conditions. The next considerations should be taken into account: first, by measuring plasma or urine PRO-C6, the cells/tissues where the existing collagen VI synthesis takes place cannot be identified and might be (partially) di fferent from the transplanted kidney. Second, PRO-C6, by definition, only measures a collagen split product of the alpha3 chain of collagen VI [15,27], whereas PSR staining is the resultant of all collagen deposits. As we know, there are >20 di fferent types of collagens, all of which can be stained by PSR [35]. So, changes in PSR staining do not necessarily correspond with changes in COL VI synthesis. Third, the PRO-C6 assay measures a split product of collagen VI that is cleaved o ff after cellular synthesis and thus reflects synthesis of collagen VI. Collagen deposition in a tissue, however, is the resultant of collagen synthesis and collagen degradation (mainly by metalloproteinases). So, the PRO-C6 assay shows one side of the coin (synthesis), whereas the other side of the coin (degradation) is not measured. We anticipate that various treatment regimens might not only influence collagen VI synthesis but collagen VI degradation as well. Next, since we did not perform immunofluorescent studies, we cannot assure that there was recurrence or enhanced interstitial inflammation; however, when stratified analyses by primary kidney disease were performed, there was no significant di fference in biomarkers or histological evidence of inflammation. Also, the possibility that incidence of glomerulonephritis was underestimated due to low use of immunofluorescence in the evaluation of biopsy materials in the regular clinical setting in which the current study was performed, and the possibility that such potential underestimation may have biased our results, is a limitation of our study. Although we performed sensitivity analyses in which we found no indication of the presence of such bias, it can, of course, not be excluded. Future studies are warranted to confirm our findings, and it would be relevant to apply immunofluorescence in such studies in order to maximize the accuracy of estimation of glomerulonephritis recurrence. It would also be interesting if future studies would compare the pre- and post-transplantation behavior of PRO-C6. Furthermore, patients receiving CsA had a more marked decline in eGFR compared to the EVL group, as was shown in the main outcomes of the MECANO publication [23]. This might have influenced both plasma and urine PRO-C6 values and di fferences between both treatment arms. Finally, we do not have information on eGFR at inclusion, therefore the eGFR changes before randomization could not be evaluated, and this prevents us from exploring the causes underlying early fibrotic lesions.

The present study has several strengths. Being a randomized clinical trial, we have a very homogenous population regarding time since transplantation and initial immunosuppressive regimen. Also, we studied PRO-C6 against the current gold standard for fibrosis detection, which is kidney biopsy [5,6], taken at the same time point as the biomarkers, allowing both cross-sectional and longitudinal analyses. Several limitations must also be considered. Most of our patients are from a European background, and care should be taken when extrapolating our findings to other ethnic groups. Also, especially at 24 months, we had a reduced number of available samples and a longer follow-up would have allowed us to further explore the prospective behavior of PRO-C6.

In conclusion, 6-month post-transplantation plasma concentration PRO-C6 has a good longitudinal association with graft biopsy-proven IFTA scores, which could make it potentially useful as a follow-up tool. On the other hand, urine PRO-C6 did not associate with fibrotic parameters measured at time of biopsy or in future protocol biopsies. Additionally, we showed a di fferential evolution of PRO-C6 during follow-up dependent on immunosuppressive regimen. For the first time, this study provides biopsy-controlled data of PRO-C6 as a potential non-invasive biomarker of graft fibrosis in KTRs. This is a first step towards non-invasive detection by plasma PRO-C6 of pro-fibrotic ECM turnover early after transplantation. The potential utility of the implementation of PRO-C6 in clinical follow-up of KTRs requires further clinical studies.t The detection of causes underlying early kidney fibrosis was not the scope of the current study, ye<sup>t</sup> we hold a plea for future studies aiming at evaluating whether primary kidney disease may influence he performance of PRO-C6 as a biomarker in KTRs. Furthermore, it would be interesting if future studies would also compare the pre- and post-transplantation behavior of PRO-C6.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-0383/9/10/3216/s1: Table S1. Biomarkers and histological characteristics during follow-up on KTRs by primary kidney disease. Table S2. Biomarkers and histological characteristics during follow-up on KTRs by primary kidney disease assuming all patients with unknown cause would have had glomerulonephritis as primary kidney disease.

**Author Contributions:** Conceptualization: C.G.S., D.G.K.R., and J.V.D.B.; Data curation: D.G.K.R., R.S.H., C.A.t.V.-K., M.v.L., M.v.D., and A.D.; Formal analysis: M.Y.-C., C.G.S., S.J.L.B., and J.V.D.B.; Investigation: D.G.K.R., R.S.H., C.A.t.V.-K., M.v.L., M.v.D., A.D., M.A.K., F.J.B., J.W.d.F., S.F., J.K., F.G., and S.J.L.B.; Methodology: S.P.B., M.A.K., F.J.B., J.W.d.F., S.F., J.K., and F.G.; Project administration: S.P.B., S.J.L.B., J.-S.S., and J.V.D.B.; Resources: D.G.K.R., M.A.K., and F.G.; Supervision: S.P.B., S.J.L.B, J.-S.S., and J.V.D.B.; Writing—original draft: M.Y.-C. and C.G.S.; Writing—review and editing: M.Y.-C., C.G.S., D.G.K.R., R.S.H, C.A.t.V.-K., M.v.L., M.v.D., A.D., S.P.B., M.A.K., F.J.B., J.W.d.F., S.F., J.K., F.G., S.J.L.B., J.-S.S., and J.V.D.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Netherlands Organization For Health Research and Development (ZonMw; gran<sup>t</sup> number 114021010, PO), the Danish Research Fund (Den Danske Forskningsfond), Novartis Pharma by means of an unrestricted grant, the Chilean National Commission of Scientific and Technological Investigation (CONICYT) (doctorate studies gran<sup>t</sup> to Sotomayor [F 72190118]) and the Graduate School of Medical Sciences of the University Medical Center Groningen (MD/PhD gran<sup>t</sup> to Hijmans).

**Conflicts of Interest:** D.G.K.R., M.A.K., and F.G. are full-time employees at Nordic Bioscience and M.A.K. and F.G. hold stocks. Nordic Bioscience is a privately-owned, small–medium-sized enterprise partly focused on the development of biomarkers and owns the patent for the ELISA used to measure PRO-C6 levels. None of the authors received fees, bonuses, or other benefits for the work described in the manuscript, and Nordic Biosicience did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. No other author declares conflict of interest.
