**4. Discussion**

In the present study, we demonstrated the clinically important improvement of blood pressure control, defined as the decline of attended o ffice systolic pressure by at least 20 mmHg without pharmacotherapeutic changes or the reduction of antihypertensive treatment due to the BP decline, in half of our cohort of HCV-infected stable kidney transplant recipients after successful HCV eradication with sofosbuvir-based DAA therapy. In this subgroup of patients, we observed the reduction of systolic BP values despite the de-escalation of antihypertensive treatment in 9 out of 14 subjects. Moreover, we also documented the parallel decline of central aortic pressure parameters, obtained with the Sphygmo-Cor device, including augmentation index, in patients with improved BP control. Finally, we noted the corresponding decrease of liver steatosis, measured by the controlled attenuation parameter, in this subgroup of patients, which may sugges<sup>t</sup> the potential underlying mechanism of beneficial vascular changes.

We are aware that nowadays a majority of dialysis patients with previously diagnosed HCV infection have already been successfully treated with DAA regimen even before they ge<sup>t</sup> a kidney transplant. Nevertheless, the confirmation of relatively moderate BP control improvement in patients being currently on dialysis would be much more di fficult due to procedural-related fluctuations in volemia. Thus, the present study findings may help to elucidate or confirm some mechanisms linking the HCV infection with BP control in CKD patients.

There are several possible pathomechanisms linking the chronic HCV infection with elevated blood pressure and increased cardiovascular risk, including increased arterial sti ffness [23], endothelial dysfunction [13], and the direct e ffect of liver steatosis on BP regulation [24]. The virus plays an etiological and pathogenic role in the development of vasculitis and renal involvement with subsequent elevated BP and cardiovascular events. Notably, in the present study, the time from HCV diagnosis to DAA treatment was significantly shorter in the subgroup with improved BP control thereafter, which may confirm that the longer duration of HCV infection determines the degree of irreversible vascular damage and, therefore, influences the net e ffect of HCV eradication on BP control. Recently, an increased augmentation index following viral eradication with DAA therapy was found in patients with advanced fibrosis (≥9.5 kPa) [25]. Of note, in patients with non-advanced fibrosis, the authors observed a stable augmentation index and significantly lower values of both o ffice and central systolic pressure at the SVR12 time point [25]. In contrast, we observed a significant decrease of the normalized augmentation index in a subgroup with BP improvement, whereas any change in arterial sti ffness, measured by pulse wave velocity, was found in our cohort, independently of BP changes after DAA treatment.

When considering the possible changes of endothelial function after successful HCV eradication with DAA, the reduction of endothelium-derived adhesion molecule levels at the SVR12 time point was confirmed by two small studies [26,27], but the improvement of FMD values was observed only in one group [26], and Davis et al. did not find changes in bedside microvascular reactivity, measured by peripheral arterial tonometry [27]. Of note, both studies were performed in individuals with normal renal function. In our cohort, we observed stable FMD values and significantly lower NMD values after DAA treatment; however, a repeated measurement with a double dose of nitroglycerin did not confirm the preceding finding. As we previously reported, the significant decrease of calcineurin inhibitor levels shortly after successful HCV eradication in this group [15], we may speculate that this e ffect was caused by an improved liver metabolic e fficiency, which limited the biologic e ffect of a standard nitroglycerin dose used originally. Furthermore, the calcineurin inhibitor co-medication may also influence the endothelial function in our group [28], though the proportion of cyclosporine A- and tacrolimus-treated patients was similar in both study subgroups.

It is important to notice that both analyzed subgroups did not di ffer at baseline in terms of demographics, co-morbidity, or kidney graft function. Also, arterial sti ffness and endothelial function measures, as well as liver function tests and liver sti ffness measurements, were comparable. There were significantly higher baseline o ffice systolic blood pressure and pulse pressure values, but not central blood pressure parameters, in subgroup 1. The posttreatment follow-up evaluation, along with the substantial BP control improvement, revealed a significant decrease of CAP values in these patients. Notably, in patients without known cause of chronic liver disease, increased CAP was shown to be a good indicator of fatty liver disease with metabolic abnormalities that manifest even before a sonographic fatty change appears [29].

In HCV-infected patients, an intrahepatic viral load directly enhances the liver steatosis [30], especially in the setting of multiple metabolic abnormalities (expansion of visceral adipose tissue, insulin resistance, reversible hypocholesterolemia, arterial hypertension, and hyperuricemia). On the other hand, in patients with non-alcoholic fatty liver disease (NAFLD), steatosis grade was the most important factor for endothelial dysfunction [31]. Furthermore, lower FMD values were shown in pediatric NAFLD patients, in whom systolic and diastolic blood pressures were significantly higher than in healthy controls, but also higher than obese children with normal livers [32]. Even more

importantly, fatty liver was shown to be associated with 24-h systolic blood pressure and daytime diastolic blood pressure measurements [24]. Recently, Wang et al. demonstrated that NAFLD is independently associated with hypertension and blood pressure category [33]. In this study, the CAP value was the predictor of diastolic, but not systolic BP in stepwise analysis for the whole study group, not only in NAFLD participants [33]. Of note, CAP alone was not sufficient for predicting hypertension in this study.

It is well known that in the population of KTRs, the measures of vascular damage are conditioned by many factors, including the duration of chronic kidney disease, pretransplant dialysis vintage, concomitant immunosuppressive regimen, and non-optimal kidney graft function. Thus, the possibility for marked improvement of vascular elasticity and distensibility after the successful HCV treatment is much lower than in the general population. Nonetheless, overall results of our investigation may indirectly confirm the notable role of decreasing liver steatosis in the BP control improvement in the study group.

Finally, FGF-21 levels are reported to increase in acute liver injury, but decrease in chronic hepatitis B patients [34], especially those with advanced fibrosis [35]. We analyzed FGF-21 concentrations before and after the successful treatment of chronic HCV infection. Its levels significantly increased only in patients with initially more advanced steatosis as shown by CAP and improved BP control after DAA therapy and HCV elimination. The concomitant decrease of liver steatosis after DAA therapy observed in our study is in line with previously published papers [36,37] and may play a significant role in improved BP control.

The main limitation of our analyzed cohort was its small size, which limited multivariate analysis of factors independently associated with the BP control improvement. Therefore, our preliminary data should be investigated in a larger multicenter cohort. However, we included all eligible KTRs with the sustained elimination of chronic HCV infection. Of note, the structure of DAA regimen in our patients was uniform: All were treated with a sofosbuvir-based therapy. Another limitation was the method of OBP measurement used in this study, specifically the lack of unattended BP measurement, which eliminated the potential bias related to the 'white coat' effect. In addition, we did not perform the 24-h ambulatory blood pressure monitoring; however, we observed the central blood pressure values and found a strong association with OBP results. Lastly, we would like to acknowledge the lack of monitoring of the adherence to antihypertensive medication in our study.
