This retrospective cohort study was conducted at the Unit of Infectious and Tropical Diseases of the Mater Domini Teaching Hospital in Catanzaro (Italy) from January 31st, 2014 to October 31st, 2016. HCV positive patients who were prescribed treatments including a DAA agent were enrolled. The study was approved by the institutional review board (IRB) of “Mater Domini” Teaching Hospital (IRB No.: 1/CE_22/01/2014) and was conducted with respect of Declaration of Helsinki. All study participants provided written consent prior to study enrollment. To be recruited, patients had to have a scheduled end of treatment at least 12 weeks before 31st October 2016 (starting analysis date).

Exclusion criteria were: i) IFN including treatment; ii) co-infection with human immunodeficiency virus (HIV); iii) hemodialysis; iv) retreatment after an interval <6 months between the sequential treatment courses; v) treatment still ongoing or with <12 weeks of available follow-up at the completion of the study; vi) hepatitis B surface antigen positivity [14].

Patients having all time-points available (baseline, end of treatment and week-12 of follow-up after the end of treatment [FU-12]) were included in the study of eGFR evolution while serum NGAL changes were evaluated in a subgroup of patients treated with SOF/ledipasvir (LDV) for which a serum sample was stored in the laboratory of the Clinical Microbiology and Virology Unit of Mater Domini Teaching Hospital of Catanzaro (Italy). Medical history, physical examination and routinely laboratory exams were performed atbaseline, end of treatment and FU-12.

Chronic Kidney Disease Epidemiology Collaboration (CKD-Epi) equation {141×min (SCr/k, 1)^α×max (SCr/k, 1)^-1.209×0.993^Age× [1.018 if female or 1.159 if black]} was used to calculate eGFR in which SCr is serum creatinine (in mg/dL), k is 0.7 for females and 0.9 for males, a is 0.329 for females and 0.411 for males, min is the minimum of SCr/k or 1, and max is the maximum of SCr/k or 1. CKD-Epi equation was preferred because is the most accurate formula for eGFR ≥60 mL/min [9,15].

Liver fibrosis was estimated at baseline by transient elastography and at each time-point either by Fibrosis-4 (FIB-4) score or aspartate transaminase (AST) to platelet ratio index (APRI). According to transient elastography, patients were considered cirrhotic when estimated liver stiffness was ≥14.5 kPa [16]. For FIB-4 calculation, the following formula was applied: age×AST/(platelets×√alanine transaminase [ALT]), in which AST and ALT were measured as IU/L, platelets as number×10⁶/μL and age was reported in years [17]. For APRI calculation, the following formula was used: ([AST/AST upper normal limit]/platelets)×100, in which AST was measured as IU/L, platelets as number×10⁶/μL and AST upper normal limit was fixed at 35 IU/L [18].

NGAL was measured at baseline, end of treatment and FU-12. NGAL assay was performed on serum samples using the BioPorto Diagnostics Human NGAL enzyme linked immunosorbent assay (ELISA) Kit 036RUO (BioPorto Diagnostics A/S, Hellerup, Denmark), for the in vitro determination of human NGAL in body fluids (e.g. plasma, serum or urine) [19]. This is a sandwich ELISA performed in 96-microwells coated with a monoclonal antibody to human NGAL. The analyte was detected with another monoclonal NGAL antibody labeled with biotin and the assay was developed by horseradish peroxidase (HRP)-conjugated streptavidin, followed by the addition of a color-forming substrate.

Serum samples and NGAL controls were diluted 1:500 in a microwell plat. Each microwell was added of 100 μL of undiluted calibrator and incubated for 60 minutes at room temperature. This step allowed NGAL binding to specific antibodies adsorbed to microwells. Unbound material was removed by using 300 μL of wash solution for three washing cycles. Successively, 100μL biotinylated monoclonal detection antibody was added to each test well and incubated for 60 minutes at room temperature. After one more washing step, 100 μL HRP-conjugated streptavidin was added to each test well and allowed to form a complex with the bound biotinylated antibody. Unbound conjugate was removed by washing. A colorforming peroxidase substrate containing tetramethylbenzidine was added to each test well. The bound HRP-streptavidin reacted with the substrate to generate a colored product. The enzymatic reaction was stopped chemically and the optical density was read at 450 nm in an ELISA reader. The absorbance was proportional to NGAL concentration in all tested samples. NGAL concentration in each sample was plotted on the validated calibration curve by a Triturus automatic analyzer. NGAL normal reference range (36-203 ng/mL) had been previously validated in our laboratory.

Patients were ranked at baseline as follows: i) KDIGO-CKD classification (stage 1 [eGFR≥90 mL/min], stage 2 [eGFR 60-89 mL/min] and stages 3a-5 [eGFR≤59 mL/min]); ii) age (≥65 years and <65 years); iii) body mass index (BMI) (normal [18.5-24.9], overweight [25-29.9] and obese patients [≥30]); iv) type of treatment (protease inhibitor [PI] including and non-PI including); v) use of ribavirin (yes and no); HCV-RNA (≥1,000,000 IU/mL and <1,000,000 IU/mL); cirrhosis (yes [transient elastography ≥14.5 kPa or clinical cirrhosis] and no [liver transient elastography <14.5 kPa]).

Quantitative variables were presented in the text as mean values and analyzed through statistical analysis based on mean regression (Student’s t-test) because we compared the same patients (without drop-out or drop-in) at the same time points. Main characteristics of the three populations (overall cohort, eGFR group and NGAL subgroup) were compared through Student’s t-test (quantitative data: eGFR, age and FIB-4) and χ² test (qualitative data: cirrhosis and gender), as appropriate.

Changes of eGFR were evaluated by comparing mean eGFR at the different time points (baseline vs. end of treatment, baseline vs. FU-12, end of treatment vs. FU-12) through paired Student’s t-test in the overall population and in patients ranked at baseline by KDIGO-CKD classification, age, BMI, type of treatment, use of ribavirin, HCV-RNA levels, and cirrhosis. A regression analysis was performed to assess eGFR variations from baseline to the end of treatment (ΔeGFR) adjusted for gender, age, use of ribavirin, basal eGFR and cirrhosis.

NGAL changes were evaluated by comparing mean serum NGAL at the different time points (baseline vs. end of treatment, baseline vs. FU-12, end of treatment vs. FU-12) through paired Student’s t - test in the subgroup of patients included in NGAL study and in the same patients ranked at baseline by use of use of ribavirin and cirrhosis. In the same subgroup of patients, also eGFR changes were evaluated by comparing mean eGFR through Student’s t-test.

In order to analyze serum NGAL trend in response to treatment, patients with serum NGAL value higher than the upper limit of normality range (36-203 ng/mL) were compared through χ² test across established time points. Linear regression analysis was performed to assess correlation of serum NGAL at each time point with: i) eGFR; ii) FIB-4; iii) APRI. Statistical significance was set at P ≤0.05.

The study design is depicted in Figure 1. Overall, 149 patients received 162 treatments including DAAs (10 patients were being treated with 2 different regimens while 1 patients was treated with four different regimens) from January 31st, 2014 to October 31st, 2016. According to the exclusion criteria, 47 treatments were removed (18 had a scheduled end of treatment less than 12 weeks before the starting analysis date or were still on course) and other 13 treatments were further excluded because of missing data (8 patients were lost to follow-up and 5 had not available eGFR at week 12 of follow-up after the end of treatment). Thus, eGFR changes were evaluated in 99 patients, accounting for 102 treatment courses. Serum NGAL changes were additionally evaluated only in 18 patients treated with SOF/LDV. Characteristics of patients at baseline of the first DAA including treatment course, characteristics and outcomes of treatments are summarized in Tables 1-3.

No statistically significant differences (P >0.05) in gender distribution, percentage of cirrhosis, mean age, mean FIB-4 score and mean eGFR value at baseline were present among the overall population (including all patients treated) and the two populations analyzed in the study (either for eGFR or NGAL analyses).

Estimated GFR changes were analyzed in 99 patients, accounting for 102 treatments. Overall, eGFR reduction was observed from baseline to FU-12 (from 86.22 mL/min to 84.3 ml/min; P =0.049) (Fig. 2). However, eGFR changes were not statistically different among patients ranked by age, BMI, type of regimen, HCV-RNA levels and cirrhosis at baseline. Interestingly, a statistically significant eGFR decrease (from 87.12 mL/min at baseline to 84.4 mL/min at FU-12; P =0.045) was observed in patients on RBV including treatment. Conversely, patients treated with regimens not including RBV did not show significant eGFR changes (from 85.17 mL/min at baseline to 83.95 mL/min at FU-12; P =0.822). According to patients ranking by KDIGO-CKD classification, eGFR was significantly reduced by the treatment only in patients with KDIGO-CKD stage 1 (from 97.99 mL/min at baseline to 94.46 mL/min at the end of treatment [P<0.001] and 93.83 mL/min at FU-12 [P < 0.001]). No statistical significant changes of eGFR were observed in patients ranked as KDIGO-CKD stage 2 and 3a-3b. At the multiple linear regression analysis, eGFR value at baseline (coeff. 0.1; SE 0.04; P=0.013) was the only variable found to be predictive of ΔeGFR from baseline to the end of treatment. No correlations were found between ΔeGFR from baseline to end of treatment and age, use of RBV, cirrhosis and gender (P >0.05). No patients experienced acute kidney injury (increase in serum creatinine of >50% above the baseline value or an increase of ≥0.3 mg/dL during last 48 hours).

Serum NGAL changes were evaluated in 18 patients and a significant increase was observed. (from 121.89 ng/mL at baseline to 204.13 ng/mL at FU-12; P=0.014) (Fig. 3A). Notably, at FU-12, 38.8% (7/18) patients had a plasmatic NGAL value higher than the normal range (36-203 ng/mL) compared with 11.1% (2/18) patients at baseline (χ²=3,704; P =0.054). By ranking patients per use of RBV, no statistically significant differences in serum NGAL were found between groups, although a serum NGAL increase was transversally observed (RBV group: from 69.33 ng/mL at baseline to 161.39 ng/mL at FU-12, P =0.075; no RBV group: from 148.16 ng/mL at baseline to 225.50 ng/mL at FU-12, P =0.301). In contrast, noncirrhotic patients showed a significant increase in serum NGAL from baseline to FU-12 (from 85.33 ng/ml to 187.85 ng/mL, P=0.021). No statistically significant differences in eGFR evolution were found (Fig. 3B).