Usefulness of morphometric image analysis with Sirius Red to assess interstitial fibrosis after renal transplantation from uncontrolled circulatory death donors

Early interstitial fibrosis (IF) correlates with long-term renal graft dysfunction, highlighting the need for accurate quantification of IF. However, the currently used Banff classification exhibits some limitations. The aim of our study was to precisely describe the progression of IF after renal transplantation using a new morphometric image analysis method relying of Sirius Red staining. The morphometric analysis we developed showed high inter-observer and intra-observer reproducibility, with ICC [95% IC] of respectively 0.75 [0.67–0.81] (n = 151) and 0.88 [0.72–0.95] (n = 21). We used this method to assess IF (mIF) during the first year after the kidney transplantation from 66 uncontrolled donors after circulatory death (uDCD). Both mIF and interstitial fibrosis (ci) according to the Banff classification significantly increased the first three months after transplantation. From M3 to M12, mIF significantly increased whereas Banff classification failed to highlight increase of ci. Moreover, mIF at M12 (p = 0.005) correlated with mean time to graft function recovery and was significantly associated with increase of creatininemia at M12 and at last follow-up. To conclude, the new morphometric image analysis method we developed, using a routine and cheap staining, may provide valuable tool to assess IF and thus to evaluate new sources of grafts.

replacement of functional renal tissue by extracellular matrix (ECM) proteins, leading to the progressive impairment of renal graft function. Among the pathological lesions observed during CAD, one of the most prominent is interstitial fibrosis and tubular atrophy (IF/TA). Other histological damages include glomerulosclerosis, splitting of glomerular capillary basement membranes and vascular intimal hyperplasia 21 . Many mechanisms which are of immunological origins or not, are involved in this multifactorial and complex process [22][23][24][25][26] . The current concept states that many processes leading to graft fibrosis, i.e. IF/TA, occur early after the transplantation, especially within the first few months 27 . IF/TA involves about 40% of kidney grafts at 3-6 months and up to 50% at 1 year, while renal function remains stable 28,29 , which suggests that renal function is a suboptimal and late marker of kidney graft dysfunction. Up to now, the only reliable method to assess IF/TA is the histological evaluation of a graft biopsy 30,31 which also allows to determine specific lesions and pathogenic processes affecting the graft 22,23 . It is also well-known that early IF correlates with long-term graft dysfunction [32][33][34] , highlighting the need for accurate quantification of IF to better identify patients requiring specific therapeutic interventions and to determine the efficacity of such interventions. IF in kidney graft is usually graded using the Banff classification 35 . However, the Banff classification exhibits some limitations. The semi-quantitative evaluation in only 4 grades (0 to 3) prevents the precise evaluation of IF evolution and may be not sensitive enough in the early stages. Furthermore, studies have shown a wide interobserver variation in the assessment of renal graft biopsies using the Banff classification 28,[36][37][38] .
The aim of the study was to assess the initial progression of interstitial fibrosis and tubular atrophy in kidney grafts from uDCD formerly classified as "Maastricht II" non heart beating donors, using a new image analysis method based on Sirius Red staining.
In the first part of our work, we will evaluate the accuracy, the robustness and reproducibility of our computerized analysis method (mIF) to assess IF. In the second part, mIF will be applied to assess IF during the first year after kidney transplantation from uDCD and mIF score will be correlated with clinico-biological data.
Baseline characteristics of uDCD (sex, age), tWIT and cold ischemia were not associated with mIF at any time. mIF at M12 (R² = 0.29, p = 0.005) but not at D0, D15-30 and M3 correlated with mean time to renal graft function recovery. In addition, mIF at M12 correlated with increase of creatininemia at M12 (R² = 0.32, p = 0.013) and at last follow-up (R² = 0.29, p = 0.005). We found the same correlation using the Banff scoring system, however, whereas there was no correlation between ci according to the Banff scoring system at M3 and creatininemia at M12 (R² = 0.15, p = 0.18), mIF at M3 tended to be associated with increase of creatininemia at M12 (R² = 0.31, p = 0.057).

Discussion
The aim of this study was to precisely describe the evolution of IF after renal transplantation with uDCD, using a new computerized image analysis method. Indeed, IF is one of the most prominent pathological lesions of CAD and correlated with renal graft prognosis 32-34 but the currently used Banff classification is a semi-quantitative system which prevents precise description of IF evolution and may not be sensitive enough in early stages. Slight but significant increase of IF on renal graft may be missed. Grimm et al. demonstrated that a precise quantification of IF by computerized image analysis provides a better surrogate marker for time to graft failure than any combination of chronic lesion scoring using the Banff schema 34 . Moreover, some studies also exhibited a wide inter-observer variation in the IF assessment according to the Banff classification 36,37 . The morphometric analysis we developed exhibited high inter-observer and intra-observer reproducibility, with ICC [95% IC] of respectively 0.79 [0.74-0.83] and 0.88 [0.72-0.95]. A variety of techniques have been used to measure renal fibrosis, each with its own advantages and disadvantages. We decided to assess IF with unpolarized Sirius red staining, firstly because Sirius red staining is commonly used in pathological laboratories since the 1980s 40 , especially to assess liver fibrosis [41][42][43][44][45] , secondly because to its high specificity for collagen fibers and its previously demonstrated superiority compared to polarized Sirius Red and trichrome staining [46][47][48][49] . Indeed, IF is typically considered to be an excess accumulation of fibrillary collagen. Types I and III collagen represent almost all collagens synthetized by fibroblasts and often predominate in fibrotic scars. Sirius red dye molecules intercalate into the tertiary groove in the structure of types I and III collagen and are strongly birefringent when observed under polarized light. Polarized Sirius Red analysis exhibited good results in IF assessment 34,49,50 but it requires individual fields to be photographed in polarized light and analyzed or cost-expensive whole slide scanners capable of polarized light. Similarly, trichrome staining, that is commonly used to the visual assessment of collagen content in the interstitium, has also be used to quantify IF by image analysis, with good results 51,52 . However, studies demonstrated than unpolarized Sirius Red had not only the best correlation with estimated glomerular filtration rate (GFR) 47,48 compared to polarized Sirius Red and trichrome staining, but also significant correlation with the decrease of GFR 33 .
In our uDCD cohort, both mIF and ci according to the Banff classification significantly increased the first three months after renal transplantation From M3 to M12, mIF significantly increased whereas Banff classification failed to highlight increase of ci. Then, mIF may be more accurate than semiquantitative Banff evaluation to identify the effects of antifibrotic therapeutic interventions. Morphometric IF tended to increase in the first month after renal transplantation but without reaching significance, due to the limited size of sample, graft biopsies being not systematically performed during the first month. However, this early and sensitive quantification of IF may be a very valuable surrogate marker to study therapeutic intervention aiming the early development if CAD in kidney transplantation. Moreover, whereas increase of creatininemia at M12 was not associated with ci Acute tubular injury (%) 63 ± 28 47 ± 22 9 ± 11 4 ± 6 <0.05
In our study, mIF at M12 was significantly associated with increase of creatininemia at M12 and at last follow-up, which confirms the need for a precise assessment of IF.
IF, which corresponds to the replacement of renal functional tissue by ECM, is not only a major concern after kidney transplantation, but also during CKD in native kidneys. Indeed, renal fibrosis still represents the final target to treat CKD 53 . Surprisingly, few studies have focused on morphometric quantification of IF on native kidneys 54,55 . Hunter et al. 54 demonstrated than high collagen matrix index and fibrillary collagen index, both assessed by quantitative morphometry after Sirius Red staining, predicted relapse and progression to ESRD during lupus nephritis. Similarly, Gibyeli Genek et al. 55 performed a quantitative evaluation of IF with Sirius Red in IgA nephritis and highlighted that such evaluation might serve as an effective novel method to determine the prognosis in IgA nephritis.
To conclude, the new morphometric image analysis method we developed, using a routine and cheap staining, may provide valuable tool to assess IF during chronic allograft dysfunction and thus to evaluate new sources of grafts. The use of this method to describe CKD on native kidney should also be evaluated.

Patients.
After institutional review board, we retrospectively included all the 66 patients who received a kidney graft from uDCD between 2007 and 2012 in Bicêtre hospital. Kidney donation followed the 2008 Declaration of Istanbul principles and the French Agence Nationale de la Biomédecine regulation. Research was approved by the committee of the Centre de Ressources Biologiques (CRB, Paris Sud University). Informed written consent was given by all the patients for the scientific use of the graft biopsies in the CRB, Paris Sud University. All procedures and the use of tissues were performed in accordance with the Declaration of Helsinki principles. Clinical reports and biological data were collected from the associated clinical database.
Histological review of kidney graft biopsies. A total of 166 kidney graft biopsies was performed in the 66 patients. The biopsies were processed for routine light microscopy, as we previously described 56 . Biopsy samples were fixed in formalin, acetic acid and alcohol (AFA), paraffin-embedded and sliced 2.5 µm thick. Slides were stained with HES (hematoxylin, eosin and saffron), Masson's trichrome (3 sections), periodic acid Schiff, Jones methenamine silver and Sirius red. Third Masson's trichrome and Sirius red staining were consecutive. Kidney graft biopsies were reviewed independently by 2 pathologists (CP and SF) for histological features according to Banff recommendations 35 in a blind manner from clinical data. Immunostaining for C4d was performed using a rabbit monoclonal A24-T anti-human C4d antibody (DB Biotech, Kosice, Slovak Republic; dilution 1/100) and a Leica BOND-MAX ™ autostainer (Leica Biosystems Newcastle Ltd, UK). Epitope retrieval was achieved using the ready-to-use Bond Epitope Retrieval Solution 1 (Leica Biosystems Newcastle Ltd, UK) after 30 min heating.

Figure 2.
Interstitial fibrosis (IF) significantly increased after renal graft transplantation. (A) According to the Banff criteria, ci remained unchanged from D0 to D15/30, increased from D0 to M3 (p < 10 -3 ) and remained stable between M3 and M12 (p = 0.37). (B) Using morphometry analysis, mIF tended to increase as early as the first month after renal transplant (p = 0.056), increase was significant from D0 to M3 (p < 10 -3 ) and remained significant from M3 to M12 (p = 0.021). Abbreviations: ci = interstitial cortical fibrosis; mIF, morphometric interstitial fibrosis; sd, standard deviation; tx, renal transplantation; *p < 0,05; ***p< 10 -3 . Figure was performed using R software 58 . www.nature.com/scientificreports www.nature.com/scientificreports/ Quantification of interstitial fibrosis by image analysis. Renal biopsy sections sliced 2.5 µm sliced and stained with Sirius red were digitalized by a ScanScope Aperio scanner (CS), using 20X objective. For each biopsy, the cortical section, defined as the part inside the renal capsule and outside the medulla, was manually selected on digital slides. Glomeruli and medium-sized arteries were deleted by the operator.
Renal-cortex fibrosis was quantified using a computer-based morphometric analysis software (Calopix, Tribvn Healthcare, Châtillon France) as previously published 56,57 . Briefly, the operator manually selects internal Sirius Red negative areas and positive areas and next runs the software that show the final selection of the Red area. The internal negative control step allows the comparison of slides that are sequentially stained in various batches of Sirius Red since staining usually may vary. Finally, the result was expressed as a percentage of the red positive area on the total cortical surface. Morphometric analyses were performed twice for each biopsy in an independant manner by two pathologists (CP and SF) who had no knowledge of the clinical data.
Statistical analyzes. Descriptive statistical methods (means, medians, standard deviations and ranges) were used to assess the distributions of variables. Wilcoxon rank sum and t tests for continuous variables, Fisher's exact and chi-squared tests for categorical variables were performed. The intraclass correlation coefficient (ICC) with its 95% CI was used to study the reproducibility of morphometric IF (mIF) measures. Correlations between quantitative variables were assessed with Pearson product-moment correlation coefficient. For all analyses, a p value <0.05 was regarded as significant. Analyses were performed using R software (version 3.2.0) 58,59 , InStat 3 software (GraphPad Software, San Diego, CA) and Prism 4 (GraphPad Software).

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.