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Diagnostic capabilities of kallistatin, IL-10 and IL-1? in patients with non-alcoholic fatty liver disease and hypertension

Results: Kallistatin levels averaged 65.03 ng/ml, 83.42 ng/ml and 111.70 ng/ml in patients with NAFLD and HT, isolated NAFLD and control group, respectively. The IL-10 level was 2.69 ng/ml and 4.90 ng/ml in patients with comorbid and isolated NAFLD, respectively, while control results averaged 8.17 ng/ml. The IL1β level in NAFLD and HT group was 4.76 pg/ml, and in isolated NAFLD group the indicator averaged 4.02 pg/ml, which exceeded the control values (0.59 pg/ml).


INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD) is the one of the most common chronic liver diseases. The prevalence of pathology in the world averages 25.2% worldwide, and the prevalence of non-alcoholic steatohepatitis (NASH), as a more severe type of NAFLD, averages 4% in the general population (1,2).
Non-alcoholic fatty liver disease is closely associated with metabolic disorders (3). Considerable attention is paid to the relationship between NAFLD and arterial hypertension (HT) that affects about one third of the world's adult population. The prevalence of HT is significantly higher in individuals with NAFLD than in the general population, and near 50% of patients with hypertension have NAFLD (4).
NAFLD can contribute to the development of hypertension, as well as occur on the background of increased blood pressure (5). It is also known that cardiovascular diseases significantly complicate the course of NAFLD (6). Therefore, diagnosis of fatty changes in the liver is especially important in the case of a comorbid combination of NAFLD and HT.
The liver biopsy is the "gold standard" for the NAFLD diagnosis. But this expensive invasive procedure is associated with a high risk of complications and false negative results in patients with NASH (7). It is the reason why a lot of studies are connected with the determine level of biomarkers to create models for steatosis and fibrosis diagnosing and predicting the course of NAFLD (8).
Cytokines play an important role in the pathogenesis of NAFLD, as NAFLD is associated with inflammatory processes in the liver (9). The variety of effects of cytokines determines their active role in the development and progression of NAFLD by stimulating hepatic inflammation, necrosis, apoptosis, and fibrosis (10).
The importance of IL-1β in the development of NAFLD has been actively studied, it is known that its activity increases in patients with NASH and correlates with the severity of fatty liver infiltration (10,11). At the same time, there is evidence that interleukin-10 (IL-10) acts as an anti-inflammatory cytokine, therefore, on the contrary, limits organ damage and prevents the progression of hepatic steatosis (12,13). Only a few studies have been performed to investigate the role of endogenous IL-10 in the progression of NAFLD (14).
Changes in cytokine balance are also observed in patients with persistently elevated blood pressure. Hypertension is thought to be associated with increased production of proinflammatory cytokines, including IL-1 (15). However, IL-10 shows protective activity under conditions of high pressure, as it counteracts the activity of the angiotensinmediated system, as well as prevents the development of vascular dysfunction (16).
Kallistatin (kallikrein-binding protein) belongs to the family of serine protease inhibitors, which are synthesized in the liver and distributed between the tissues of the heart and blood vessels (17).
This protein maintains the level of blood pressure (BP) and exhibits anti-inflammatory, antioxidant, antiangiogenesis and antitumor effects (18,19). Various studies confirm that kallistatin may be an effective biomarker for detection of liver fibrosis in various liver diseases (20,21).
Different studies suggest that patients with NAFLD have an imbalance of pro-inflammatory and anti-inflammatory cytokines (22). Therefore, the determination of the activity of biomarkers can play a significant role in diagnosing the impact of concomitant hypertension on the course of NAFLD and determining the optimal management of this category of patients.
The aim of the study to determine the role of kallistatin, IL-1β and IL-10 as diagnostic biomarkers of NAFLD severity in patients with concomitant HT.

MATERIALS AND METHODS
The study was approved by the Ethics and Bioethics Commission of institution in which the study was conducted. The experimental part of this study respects the ethical standards in the Helsinki Declaration of 1975, as well as the principles of Good Clinical Practice (GCP) and the national law. All patients voluntarily decided to participate in the study and signed the patient's informed consent.
We examined 115 patients with NAFLD at the stage of nonalcoholic steatohepatitis (NASH). Participants included 57 men and 58 women aged 38 to 59 years (Mean=48,4). The patients were divided into two groups depending on the presence of concomitant HT. The main group (n=63) consisted of 32 men and 31 women aged 38 to 59 years (Mean=48,4) with NAFLD and HT, and the comparison group (n=52) included 25 men and 27 women aged 39 to 59 years (Mean=48,3) with an isolated course of NAFLD. NAFLD and HT I stage were observed in 21 patients (12 men and 9 women; aged 39 to 57 years (Mean=46, 9), and NAFLD and HT II stage were in 42 patients (20 men and 22 women; aged 38 to 59 (Mean=46, 9)).
The control group (n=20) consisted of practically healthy 12 women and 8 men aged 38 to 56 years (Mean=47,1), randomized by age and sex. The study of anthropometric parameters included measuring height in centimeters using a height meter, as well as body weight in kilograms using an electronic weigh scale. Body mass index (BMI) was calculated according to generally accepted formula: weight (in kilograms) divided by height (in meters) squared.Biochemical parameters of liver functional activity were determined by spectrophotometric and colometric methods (including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT)).
All statistical processing of obtained results was performed using computer software packages "Excel 2019" (Microsoft), "Statistica 8.0. for Windows" (StatSoft Inc.). Continuous variables are presented as mean (M) or median (Me) depending on the correspondence with the normal distribution and confidence intervals with established reliability γ=0,95 (95% CI) and coefficient of variation (CV) expressed as a percentage. With CV≤10%, the sample was considered weakly variable, with CV ranging from 10% to 20%, the sample was considered moderate variable, with CV≥20%, the sample was considered highly variable.
Determination of the statistical significance of differences in relative indicators was performed using Pearson's chisquared test. The Mann-Whitney U-test was used to determine the difference between the two independent samples by the trait level. Spearman's rank correlation coefficient was determined and Evans's scale was used to determine the strength of the relationship. The maximum allowable probability of committing a type I error (p-value) was established as the value of the level of statistical significance less or equal than 0,05.

RESULTS
There were no significant differences between anthropometric parameters of patients with NAFLD and HT comorbidity and isolated NAFLD. At the same time, significant differences were found in comparison with the control group in almost all indicators of both the main and the comparison group. It was expected that blood pressure values differed significantly between groups (Table 1).
Among the indicators of the functional state of the liver, the levels of hepatic transaminases had exceeded normal values and were significantly higher in patients with NAFLD and HT compared with the group of isolated NAFLD. Alkaline phosphatase and gamma-glutamyl transpeptidase were predominantly within the normal range, but in the group with comorbid NAFLD its levels were statistically significantly higher than in the group with isolated NAFLD and in the control group ( Table 1).
Kallistatin levels were decreased in patients with increasing body mass index (BMI) both in the group with NAFLD and HT (r=-0,58; p<0,001) and in the group with isolated NAFLD (r=-0,54; p=0,002).With an increase in body weight, the activity of IL-1β also increased (r=0,32) and the strength of the anti-inflammatory protection of IL-10 (r=-0,27) decreased in the group with comorbidity of NAFLD and HT.
The activity of kallistatin decreased with the progression of HT in patients with NAFLD (p<0,001). The correlations between kallistatin and CRP were very strong (r=-0,89) and strong (r=-0,61) in groups with comorbid and isolated NAFLD, respectively. At the same time, an inverse relationship between the level of CRP and IL-10 was observed in both groups of patients with NAFLD, but in cases with concomitant HT, the strength of the relationship was significantly higher (r=-0,69 and r=-0,30, respectively). In addition, in patients with NAFLD and HT with higher CRP levels, the activity of the proinflammatory cytokine IL-1β increased (r=0,61).
We also obtained data on the inverse relationship between kallistatin and hepatic transaminases in both groups of patients with NAFLD, while in cases with concomitant HT, the correlations were stronger. Also, an inverse relationship between the level of kallistatin and ALPwas found in all patients with NAFLD, but inverse correlations between the kallistatin level and GGTP (r=-0,43) was found only in the group with NAFLD and HT (Table 4).

DISCUSSION
The effect of concomitant hypertension on the clinical manifestation of NAFLD has been determined in various studies. Ampuero J. et al. showed that HT was independently linked to significant fibrosis in patients with NAFLD (23). The possibility of the two-way relationship between these diseases remains under study. Prospective cohort study of Liu P. et.al. also showed bidirectional association between NAFLD and hypertension was found (24).
The study of Ma J. showed that NAFLD is associated with increased odds of incident HT and patients with hypertension at baseline had higher odds of NAFLD development (25). According to Aneni E. C. et al. study results more prevalent NAFLD may occur early in the development of HT, even in conditions of the absence of other metabolic risk factors. Authors concluded that controlling blood pressure levels among even non-obese hypertensive patients may be important in preventing or limiting NAFLD (26).
In different studies high hs-CRP values was found in hypertensive patients and it was considered as an independent risk factor for HT. Authors concluded that this biomarker can aggravate hypertension by participating in local and systemic inflammatory responses (27). Significant differences in the CRP level obtained during the examination of patients with comorbid and isolated NAFLD course also confirm this statement.Summary from 19 prospective studies by Lonardo A. at al. suggest that HT can lead to differences in NAFLD course and condition the rapid deterioration of fatty liver patients and combination of this pathologies could be a precursor of the metabolic syndrome (28).
Various studies suggest that kallistatin concentrations vary in different chronic liver diseases, which may be associated with decreased hepatic protein secretion activity (4). Cheng Z. et al. found a significantly lower content of kallistatin in patients with liver fibrosis (29). Halla M. et al. proved that even a single determination of the biomarker level allows to detect patients in the initial liver fibrosis stages with a sensitivity of 96,7% and a specificity of 50% (7).
Frühbeck, G. et al. proved that kallistatin has a potential protective role in the obesity development in NAFLD (30). In our study, significant changes in kallistatin levels with weight gain in NAFLD patients with and without HT were detected. This may indicate the importance of the protein in protecting against metabolic changes in adipose tissue in NAFLD, but these findings require further investigation.
Another important issue is the kallistatin role in the cardiovascular diseases. The hypertension development is associated with activity of the kallikrein-kinin system decrease. Studies show that endogenous kallistatin is a protective agent against vascular oxidative stress, inflammation and fibrosis in animal hypertension models (31). The results of our study showed that kallistatin levels are significantly lower in patients with NAFLD and HT than in isolated NAFLD patients (p<0,001).
However, literature data indicate a relatively low sensitivity and specificity of kallistatin as an isolated indicator of liver parenchyma changes (21). Thus, it is necessary to consider the possibility of combining the kallistatin determination with other non-invasive NAFLD tests. Our study showed that the level of anti-inflammatory cytokines (interleukin-10) in patients with isolated NAFLD was significantly higher than in patients with a combination of NAFLD and HT, and in the control group the anti-inflammatory activity was significantly higher than in patients with NAFLD. Meanwhile, the level of IL-1β in patients with concomitant pathology of NAFLD on the background of HT significantly exceeded the corresponding value in the group with isolated NAFLD, and in general the level of inflammatory marker in patients of the main group and the comparison group exceeded control values.
These results confirm the findings of Nelson J. and other authors that proinflammatory cytokines, such as IL-1β, are elevated in nonalcoholic steatohepatitis (32). Statistically significant differences were found between kallistatin and IL-1β levels in patients with NAFLD on the background of HT depending on the stage of hypertension. We also obtained data indicating the effect of the progression of hypertension on the proinflammatory activity of IL-1β, as well as a significant inverse dependence of the antiinflammatory activity of calistatin on blood pressure. These results indicate a deepening of pro-inflammatory and antiinflammatory deviations in patients with comorbid NAFLD in the transition to more severe stages of HT and with an increase in BP grade. Also noteworthy was the fact that a significant decrease in the anti-inflammatory activity of kallistatin with increasing BMI was observed in all patients with NAFLD, and an increase in the effect of interleukins on the course of NAFLD with increased BMI was determined only under the additional influence of HT.
The lack of a clear significant relationships between IL-10 and clinical and laboratory parameters of the examined patients partially confirms the opinion of the authors that this cytokine has different effects. Thus, Saraiva M. and coauthors claim that its antifibrotic activity is detected only at the beginning of the disease, and with the progression of NAFLD the cytokine may even stimulate irreversible changes in the liver parenchyma (33). Therefore, it is important to measure the levels of these biomarkers in combination to diagnose the severity of NAFLD more accurately and to determine the effect of hypertension on the course of NAFLD.

CONCLUSIONS
The course of both isolated NAFLD and comorbidity NAFLD with HT was accompanied by significant changes of kallistatin, IL-10 and IL-1β levels, which confirmed the pathophysiological role of chronic low-grade systemic inflammation in NAFLD. The combination of NAFLD and HT led to a significant increase in the severity of deviations in these indicators, which makes it possible to consider HT as a trigger factor of NAFLD progression.
Higher HT stage and BP grade, increased BMI and high CRP levels are associated with significantly more pronounced deviations of these indicators and should be considered as additional factors of NAFLD progression in patients with HT.
The obtained data provide the possibility to consider the kallistatin, IL-10 and IL-1β as biomarkers of NAFLD severity