**3. Results**

#### *3.1. Study Group*

Out of all 73 KTRs with the presence of anti-HCV antibodies, HCV-RNA was detected in 40 patients. These patients were qualified to further HCV genotyping and viral load. Out of them, 8 patients had started DAA therapy without baseline examination planned for the present study and were not included in the final analysis. The other 32 patients were treated with the DAA anti-HCV protocol, including 8 with a 6-month regimen and 24 patients with a 3-month regimen based on sofosbuvir. The DAA regimen was shortened in the later enrolled patients as the treatment recommendations were updated during the study period. Out of this group, 4 KTRs were excluded: 2 of them were normotensive prior to HCV treatment and 2 others did not finalize the study protocol (Figure 1). Hence, the final study group consisted of 28 hypertensive patients who completed DAA therapy and both baseline and follow-up examinations. In all study patients, the diagnosis of HCV infection was established prior to kidney transplantation. In all

patients, HCV viremia was not detectable after the first month of treatment and they all reached sustained virologic response at 48 weeks from DAA treatment start (SVR48) time point. The clinical characteristics of the study patients are presented in Table 1.

**Figure 1.** Study flow chart. KTRs, kidney transplant recipients; HCV, hepatitis C virus; PCR, polymerase chain reaction.



## *p* < 0.01 versus baseline. CI, confidence interval; IQR, interquartile range; OBP, attended office blood pressure; DAA, direct antiviral drug therapy; BMI, body mass index; eGFR, estimated glomerular filtration rate; KTx, kidney transplantation; CyA, cyclosporine A; Tc, tacrolimus.

#### *3.2. Study Subgroups Based on Blood Pressure Control*

In the follow-up period, half of the patients showed an improvement of OBP control (subgroup 1). Patients with OBP improvement had initially higher systolic BP (SBP) (*p* = 0.02), but similar diastolic BP (DBP) (Table 2). In addition, they received more antihypertensive medications (mean: 2.5 vs. 1.9 drugs) before the start of DAA therapy; however, this di fference was not statistically significant. The observed overall SBP ( Δ −20.4, 95% CI, −26.2 to −14.6 mmHg) and DBP ( Δ −12.5, 95% CI, −16.5 to −8.5 mmHg) decline in subgroup 1 was obtained despite the reduction in the number of antihypertensive drugs in 9 subjects. We also observed mild reduction in SBP ( Δ −5.2, 95 % CI, −9.7 to −0.8 mmHg) and DBP (Δ −4.6, 95% CI, −9.6 to 0.3 mmHg) in the second subgroup.

At baseline, the antihypertensive treatment was used in all patients: Beta-blockers in 67.9%, calcium channel blockers in 25%, angiotensin-converting enzyme inhibitor or angiotensin receptor blocker in 32.1%, alpha-blocker in 25%, and diuretics in 32.1% of study participants. The structure of antihypertensive medication classes was similar in both study subgroups (data not shown).

Both study subgroups did not di ffer in respect to age, gender, BMI, pretransplant dialysis vintage, time after kidney transplantation, and the occurrence of diabetes (Table 1). The time from diagnosis of HCV infection to DAA treatment was significantly longer in the subgroup 2 (Table 1), however, the percentage of patients with HCV infection lasting more than 13.7 years (mean value in the whole group) did not di ffer between study subgroups (*p* = 0.13). Out of whole group, only 4 patients were previously treated with interferon-based anti-HCV regimens. There were no di fferences in regards to baseline values of serum lipid concentrations, fasting glucose and insulin concentrations, glycated hemoglobin, and HOMA-IR values between subgroups (Supplementary Table S1).

Also, the HCV genotypes were similar in both subgroups, including 11 patients with genotype 1b in each group. The percentage of patients with advanced fibrosis, defined based on a METAVIR score >2, was comparable (28.6 vs. 38.5%, *p* = 0.59). The mean time between baseline and follow-up study examinations was also similar (15.0 ± 1.4 vs. 15.9 ± 2.1 months, *p* = 0.22).

Both study subgroups were similar in respect to calcineurin inhibitor (CNI) structure (Table 1). There were no CNI-type conversions during the whole study period. At baseline, median cyclosporine (CyA) doses were comparable (100 (100–125) mg in subgroup 1 vs. 125 (100–150) mg in subgroup 2, *p* = 0.60), whereas median tacrolimus (Tc) doses were significantly greater in subgroup 2 (4.0 (2.5–6.0) mg vs. 1.0 (1.0–2.0) mg in subgroup 1, *p* < 0.05). Notably, median CyA (97 (70–128) vs. 125 (100–146) ng/mL, respectively; *p* = 0.30) and Tc (7.1 (6.1–7.4) vs. 8.4 (6.7–8.7) ng/mL, respectively; *p* = 0.22) blood trough concentrations were similar. During and after DAA treatment, the improved liver function resulted in the reduction of calcineurin inhibitor (cyclosporine or tacrolimus) blood trough concentrations as compared with baseline values, which were similar in both study subgroups (−21.4 (−37.2 to −5.7) vs. −11.3 (−33.5 to 10.9)%, respectively; *p* = 0.42) and required individual dose adjustments in 61% of patients (*n* = 17) as soon as after one month of therapy. The consecutive CNI dose adjustments were made at physician discretion and were guided by the drug blood concentration, to prevent the CyA level decreasing below 70 ng/mL or Tc level decreasing below 5 ng/mL. Overall, the median CNI dose changes in both study subgroups were similar (13.4 (interquartile range (IQR) 0–25) vs. 25 (0–75)%, respectively; *p* = 0.26). Also, the absolute median dose changes of CyA (0 (0–75) vs. 25 (12.5–25) mg, respectively; *p* = 0.73) and Tc (0.5 (0.5–1.0) vs. 0 (−1.5–0.5) mg, respectively; *p* = 0.26) were similar.


#### *J. Clin. Med.* **2020**, *9*, 948

#### *3.3. Liver Function Tests and Liver Morphologic Assessments*

At baseline, there was a numerical difference in HCV viremia between subgroups (Table 1), but neither HCV viremia nor baseline liver function tests differed significantly. In both subgroups, there was a significant reduction in aminotransferases and GGT activities after DAA treatment (Supplemental Materials Table S2). Liver elastography measurements were performed with a success rate near 100% (only 3 out of 28 patients needed 11 total measurements to obtain 10 valid results). Baseline liver stiffness measurements did not differ between subgroups and remained unchanged thereafter. On the contrary, CAP values only declined significantly in subgroup 1 (Supplementary Materials Table S2 and Figure 2).

**Figure 2.** Individual plot of the controlled attenuation parameter at baseline and after the follow-up in patients with and without attended office blood pressure control improvement.

#### *3.4. Central Blood Pressure Parameters*

Both baseline and follow-up values of central aortic systolic pressure and central aortic pulse pressure correlated with corresponding values of OBP (baseline *r* = 0.669, *p* < 0.001, follow-up *r* = 0.557, *p* < 0.01 for SBP; and baseline *r* = 0.730, *p* < 0.001, follow-up *r* = 0.502, *p* < 0.01 for pulse pressure, respectively). There were also significant correlations between baseline augmentation index values standardized to heart rate (AIx@75) and both baseline and follow-up values of office SBP and pulse pressure (baseline *r* = 0.660, *p* < 0.001 and *r* = 0.562, *p* < 0.01, respectively; follow-up *r* = 0.484, *p* < 0.01 and *r* = 0.545, *p* < 0.01, respectively) (Table 3).

Parallel to OBP, we observed a 15.4% decline in aortic systolic pressure, a 12.1% decline in aortic diastolic pressure, and a 21.7% decline in aortic pulse pressure in subgroup 1 after successful DAA treatment (Table 3). Of note, AIx@75 values decreased significantly only in subgroup 1. On the contrary, only a mild decline of systolic aortic pressure (7.2%) was observed in subgroup 2. There was a positive correlation between the percentage change in CAP values and both the change in central aortic systolic pressure (*r* = 0.438, *p* < 0.05) and the change in AIx@75 (*r* = 0.446, *p* < 0.05). Of note, both baseline (*r* = 0.479, *p* < 0.05) and follow-up (*r* = 0.431, *p* < 0.05) CAP results correlated with corresponding BMI values.



values between two study subgroups, *p* values were >0.05.

#### *3.5. Arterial Structural and Functional Measurements*

At baseline and in the follow-up period, PWV values were similar in both subgroups (Table 3). FMD values were stable in both subgroups, while NMD measured using the standard (400 μg) dose of nitroglycerin did not change in subgroup 1, but showed a significant reduction in subgroup 2 (Table 3). However, in an additional repeated NMD measurement with a double dose of nitroglycerin there was no significant NMD change compared with baseline values in both study subgroups.

#### *3.6. Cardiac Parameters*

At baseline, both mean LVM (210 (169–250) vs. 211 (173–250) g, *p* = 0.84)) and mean LVMI (113 (94–131) vs. 114 (95–133) g/m2, *p* = 0.98) were comparable in subgroups 1 and 2, respectively. Similarly, there were no di fferences in the follow-up LVM and LVMI values (0.70 and 0.87, respectively), and their absolute changes during the study period did not di ffer significantly (LVM 7.9 (−23.5 to 39.2) vs. −0.3 (−44.4 to 43.8), *p* = 0.57; LVMI 3.3 (−13.4 to 19.9) vs. −1.3 (−23.2 to 20.6) g/m2, *p* = 0.70).

#### *3.7. Inflammatory Markers*

The baseline median of CRP concentration was slightly higher in subgroup 1 (1.7 (IQR 1.1–2.4) vs. 0.9 (0.4–1.4) mg/<sup>L</sup> in subgroup 2, with borderline significance (*p* = 0.07)). At follow-up assessment, there was a borderline increase of median hsCRP in subgroup 2 (3.6 (0.5–6.7), *p* = 0.06), whereas no di fference was noted in subgroup 1 (1.8 (1.5–3.0), *p* = 0.33). There were no significant di fferences in IL-6 concentration median values, both at baseline (2.7 (2.2–3.2) vs. 2.2 (1.8–2.5) pg/mL, *p* = 0.23) and at the follow-up examination (2.7 (1.7–4.1) vs. 2.6 (1.7–5.7) pg/mL, *p* = 0.76). Levels did not change significantly during the study period.

#### *3.8. Fibroblast Growth Factor 21 Levels*

Median values of FGF-21 measured at follow-up increased significantly in subgroup 1 (411 (204–706) vs. 215 (120–535) pg/mL at baseline, *p* < 0.01), while they remained unchanged in subgroup 2 (189 (147–481) vs. 226 (116–267) pg/mL at baseline, *p* = 0.22). The baseline number of antihypertensive drugs was borderline associated with log values of baseline FGF-21 (*r* = 0.375, *p* = 0.05) and significantly associated with follow-up values of FGF-21 (*r* = 0.467, *p* < 0.05). However, the log values of both baseline and follow-up FGF-21 did not correlate with baseline SBP or DBP values, whereas they correlated significantly with baseline central systolic aortic pressure (*r* = 0.388, *p* < 0.05, and *r* = 0.434, *p* < 0.05, for baseline and follow-up FGF-21 log values, respectively) and borderline with central diastolic aortic pressure (*r* = 0.357, *p* < 0.05, and *r* = 0.385, *p* < 0.05, respectively). Moreover, the log values of follow-up FGF-21 negatively correlated with the change of central systolic and diastolic aortic pressures (*r* = −0.361 and *r* = −0.367, respectively) with both *p* values = 0.06.
