**3. Results**

#### *3.1. Baseline Characteristics*

The characteristics at enrollment and at randomization of a total of 94 patients, 51 in the CsA group and 43 in the EVL group, are displayed in Tables 1 and 2. At enrollment, in the overall population, the mean (*SD*) age was 52 (13) years-old, and most patients were male and Caucasian. The main cause of end-stage kidney disease in this trial was polycystic kidney disease (24%), followed by glomerulonephritis (17%) and hypertension (16%). The mean donor age was 50 (13) years old, and the most frequent type of donors was living unrelated (31%), followed closely by deceased after brain death (30%). The median antigen mismatch was 3, and the median (IQR) of total time on kidney replacement therapy was 24 (5–46) months.

At randomization, 6 months after the beginning of the trial, patients had a mean graft function, as assessed by the estimated glomerular filtration rate (eGFR), of 49 (42–62) mL/min/1.73 m2. Patients had a mean weight of 79 (14) kg and a mean systolic blood pressure of 144 (20) mmHg. Mean low-density lipoprotein (LDL) was 3.19 (2.39–3.75) mmol/L, and 59% of patients were statin users. Mean glycated hemoglobin was 6.08% (1.10), and only two patients had the diagnosis of diabetes mellitus. Fifteen patients (16%) were active smokers. Concerning subgroup differences, patients in the CsA group had an apparent higher weight (81 vs. 78 kg), a more frequent use of statins (63% vs. 54%), and a higher percentage were active smokers (20% vs. 12%) when compared to the EVL group. Also, the two diabetic patients were both in the EVL group. None of these differences was of statistical significance.


**Table 1.** Characteristics at enrollment of study population, overall kidney transplant recipients (KTRs), and randomization groups.

a Data available in 87 patients. CsA: cyclosporine A; EVL: everolimus; TTKRT: total time on kidney replacement therapy.

**Table 2.** Characteristics at randomization of study population, overall KTRs, and randomization groups.


Data available in a 90, b 92, and c 88 patients. eGFR: estimated glomerular filtration rate; BMI: body mass index; SPB: systolic blood pressure; DBP: diastolic blood pressure; LDL: low-density lipoprotein; HDL: high-density lipoprotein; HbA1c: glycated hemoglobin.

#### *3.2. PRO-C6 and Biopsy-Proven Histological Changes over Follow-Up*

Mean (*SD*) plasma PRO-C6 at 6 and 24 months was 9.5 (3.4) and 9.4 (4.3) ng/mL, respectively, without significant di fferences between the two groups. As for urine, median (IQR) PRO-C6 at 6 and 24 months after correction by creatinine was 6.7 (4.8–12.4) and 5.9 (3.4–21.5) ng/mg, respectively. Plasma and urine PRO-C6 did not correlate at either 6 or 24 months (Spearman's ρ 0.226, *p* = 0.09; Spearman's ρ 0.311, *p* = 0.11; respectively). No di fference in urine PRO-C6 between the two study groups was

present at 6 months, but at 24 months mean urine PRO-C6 was significantly higher in the EVL group compared to the CsA group (7.5 vs. 4.5 ng/mg; *p* = 0.02). Delta plasma PRO-C6 was positive in both subgroups and was not significantly different. As for delta urine PRO-C6, it was positive in the EVL group and negative in the CsA group; this difference was statistically significant (0.9 vs. −1.4 ng/mg; *p* = 0.01). (Table 3).


**Table 3.** Biomarkers and histological characteristics during follow-up of overall KTRs and randomization groups.

Data available in a 73, b 62, c 36 patients. CsA: cyclosporine group; EVL: everolimus group; PRO-C6: released pro-peptide of collagen type VI (endotrophin); IF/TA: interstitial fibrosis/tubular atrophy; PSR: Picro Sirius Red; ti-score: total inflammation score.

Histological analyses at 6 months showed a median IF/TA score of 1 (0–1) points and a mean PSR staining percentage of 13.3% (6.0), with no significant differences between patients in the CsA and EVL groups. Inflammation, as evaluated by the ti-score, was also not significantly different between the two groups. At 24 months, the overall population showed a higher IF/TA score, PSR percentage, and ti-score when compared to the previous biopsy. At this time point, the PSR staining percentage was higher in the CsA group compared to the EVL group (19.7% vs. 14.5%; *p* = 0.02); no significant difference was present in the other histological parameters (Table 3).

When patients were stratified by their primary kidney disease, no significant differences were found in the plasma and urine concentrations of PRO-C6 at any time point during follow-up. No significant difference was found either in fibrosis (IF/TA and PRO-C6) or inflammation (ti-score) at 6 and 24 months (Table S1). Also, no significant differences were found in sensitivity analyses in which all KTRs with unknown cause of primary kidney disease were considered as patients with glomerulonephritis as primary kidney disease (Table S2).

#### *3.3. Association between PRO-C6 and Biopsy Changes*

Plasma PRO-C6 at 6 months post transplantation was significantly associated with 6-month and 24-month IF/TA scores (Std. β = 0.34 and 0.24, respectively; both *p* < 0.05). A prospective association was also present for 6-month plasma PRO-C6 with 24-month biopsy proven inflammation (ti-score) and the delta inflammation between the two biopsies (Std. β = 0.23 and 0.22, respectively; both *p* < 0.05). No association was found between 6-month plasma PRO-C6 and 6- or 24-month PSR. Also, no cross-sectional association was found between 24-month plasma PRO-C6 and histological evidence of fibrosis or inflammation. Urine PRO-C6 at 6 months only showed a prospective and inverse association with 24-month PSR (Std. β = −0.30; *p* < 0.05), and there were no cross-sectional associations at 24 months. Delta plasma and urine PRO-C6 did not correlate with either histological evidence of fibrosis or inflammation (Table 4).



\* *p* value < 0.05; \*\* *p* value < 0.01. Linear regression analyses were performed. Std. β coefficients represent the difference (in standard deviations) in each biomarker per 1 standard deviation increment in each individual biopsy score. PRO-C6: pro-peptide of collagen VI (endotrophin); Std. β: standardized beta coefficient; IF/TA: interstitial fibrosis/tubular atrophy; PSR: Picro Sirius Red; ti-score: total inflammation score.

When patients were divided by randomization group, no significant associations were found between 24-month plasma PRO-C6 and histological changes. Urine PRO-C6 was significantly and inversely associated with the delta of IF/TA score in patients among the CsA group, and no other significant association was found. Delta plasma and urine PRO-C6 were not significantly associated with any histological changes (Table 5).



\* *p* value < 0.05. Linear regression analyses were performed. Std. β coefficients represent the difference (in standard deviations) in each biomarker per 1 standard deviation increment in each individual biopsy score. PRO-C6: pro-peptide of collagen type VI (endotrophin); CsA: cyclosporine group; EVL: everolimus group; Std. β: standardized beta coefficient; IF/TA: interstitial fibrosis/tubular atrophy; PSR: Picro Sirius Red; ti-score: total inflammation score.
