CHANGES IN EXTRACELLULAR MATRIX COMPONENTS METABOLISM IN PATIENTS WITH NONALCOHOLIC STEATOHEPATITIS ON THE BACKGROUND OF OBESITY AND COMORBIDITY WITH CHRONIC KIDNEY DISEASE

The purpose of the study was to find out the features of the state of carbohydrate-protein components of the connective tissue of the liver and kidneys extracellular matrix in non-alcoholic steatohepatitis in patients with obesity of the 1 degree and chronic kidney disease of the І–ІІ stages. We examined 140 patients with non-alcoholic steatohepatitis with comorbid obesity of the 1 degree and chronic kidney disease of the I–II stages. Patients were divided into 2 groups that were randomized according to age, sex, degree of obesity, and stage of chronic kidney disease (chronic uncomplicated pyelonephritis with latent course in the phase of retinal exacerbation). The control group consisted of 30 practically healthy persons of the corresponding age and sex. As a result of studies, it was found out that a significant increase in the synthesis of collagen and glycosaminoglycans was observed in patients with non -alcoholic steatohepatitis that arose on the background of obesity. It was accompanied by ineffective resorption of newly formed collagen due to inhibition of the collagenolytic activity of blood plasma, due to significant activation of proteinase inhibitors, significant imbalance in the system metabolism of connective tissue. Under the conditions of comorbidity of nonalcoholic steatohepatitis and chronic kidney disease, the synthesis and resorption of collagen are activated. In spite of compensatory activation of collagenolysis, the anabolism processes predominated with a significant hyperproduction of actinic-phase proteins, fibronectin, glycosaminoglycans, fibroblast growth factor, and led to progressive fibrosis of the liver and disturbance of its functions.

The purpose of the study was to find out the features of the state of carbohydrate-protein components of the connective tissue of the liver and kidneys extracellular matrix in non-alcoholic steatohepatitis in patients with obesity of the 1 st degree and chronic kidney disease of the І-ІІ stages.
We examined 140 patients with non-alcoholic steatohepatitis with comorbid obesity of the 1 st degree and chronic kidney disease of the I-II stages.Patients were divided into 2 groups that were randomized according to age, sex, degree of obesity, and stage of chronic kidney disease (chronic uncomplicated pyelonephritis with latent course in the phase of retinal exacerbation).The control group consisted of 30 practically healthy persons of the corresponding age and sex.
As a result of studies, it was found out that a significant increase in the synthesis of collagen and glycosaminoglycans was observed in patients with non -alcoholic steatohepatitis that arose on the background of obesity.It was accompanied by ineffective resorption of newly formed collagen due to inhibition of the collagenolytic activity of blood plasma, due to significant activation of proteinase inhibitors, significant imbalance in the system metabolism of connective tissue.
Under the conditions of comorbidity of nonalcoholic steatohepatitis and chronic kidney disease, the synthesis and resorption of collagen are activated.In spite of compensatory activation of collagenolysis, the anabolism processes predominated with a significant hyperproduction of actinic-phase proteins, fibronectin, glycosaminoglycans, fibroblast growth factor, and led to progressive fibrosis of the liver and disturbance of its functions.
Relationship of work with scientific programs, plans, themes.This work is a fragment of the re-search work «Pathogenetic mechanisms of mutual burden and clinical features of the course of nonalcoholic fatty liver disease and chronic kidney disease, justification of differentiated treatment», state registration number is 0111U006303.
Introduction.The comorbidity of non-alcoholic steatohepatitis (NASH) and chronic kidney disease (CKD) on the background of obesity has recently drawn the attention of both practitioners and researchers [1,2].An important role in the pathogenesis of progression of liver and kidney diseases is played by the system of components of connective tissue (CT) of the extracellular matrix (PCM) [3,4,7].According to the literature, non-alcoholic fatty liver disease (NAFLD) in progress leads to the development of both liver cirrhosis and hepatocellular carcinoma, the incidence of which on the background of NAFLD substantially exceeds the indicators in the population.Scientists frequently attempted to find new probable biochemical markers of fibrosis formation intensity [8,9,11], to increase the diagnostic value, sensitivity and specificity of existing methods, and to develop methods of influence to inhibit these processes.
The purpose of the study was to find out the features of the state of carbohydrate-protein components of the connective tissue of the liver and kidneys extracellular matrix in non-alcoholic steatohepatitis in patients with obesity of the 1 st degree and chronic kidney disease of the І-ІІ stages.
Material and methods of research.We examined 140 patients with non-alcoholic steatohepatitis (NASH) with comorbid obesity of 1 st degree and chronic kidney disease (CKD) of І-ІІ stages.Patients were divided into 2 groups that were randomized according to age, sex, degree of obesity, and stage of chronic kidney disease (chronic uncomplicated pyelonephritis with latent course in the phase of retinal exacerbation).The first group comprised 58 patients with NASH on the background of obesity (without accompanying CKD), the second group had 52 patients Український журнал медицини, біології та спорту -Том 3, № 7 (16) with NASH on the background of obesity with a comorbid CKD I-II stages.The control group consisted of 30 practically healthy persons of the corresponding age and sex.
Changes in the metabolism of the components of the extracellular matrix were determined by the free oxyproline content in blood (FOP) by S. S. Tetyanets (1985) and protein-bound oxyproline (PBOP) by M. S. Osadchuk (1979), hexosamines (HA) by O. G. Archipova (1988), seromucoid (SM), sialic acid (SA), fucose-free protein (FFP), using Danush Ltd (Lviv), ceruloplasmin (CP) by the Revina method (1976), the level of collagenolytic activity of blood plasma (CLA): according to the intensity of azocel lysis; the content of the fibroblast growth factor (FGF) in the blood, and also on the parameters of the total fibrotest (T.Pounard) by the enzyme-linked immunosorbent assay (ELISA).
The diagnosis of NASH was established in accordance with the unified clinical protocol, approved by the order of the Ministry of Health of Ukraine No. 826 dated 06.11.2014, in the presence of criteria for the exclusion of chronic diffuse liver disease of the viral, hereditary, autoimmune or medicinal genesis as causes of cholestatic or cytolytic syndromes, as well as the results of the USG survey.Diagnosis and treatment of CKD were performed according to the recommendations of the clinical guidelines of the State Institute «Institute of Nephrology, NAMS of Ukraine» (2012).
The statistical analysis of the results was carried out in accordance with the type of research carried out and the types of numerical data that were obtained.Distribution normality was verified using Liliefors, Shapiro-Uilka tests and the direct visual evaluation of eigenvalues distribution histograms.Quantitative indices having a normal distribution are represented as mean (M) ± standard deviation (S).Discrete values are presented in the form of absolute and relative frequencies (percentage of observations to the total number of surveyed).We used parametric tests to estimate the Student's t-criterion, Fisher's F-criterion and to compare the data with a normal distribution pattern.In the case of abnormal distribution, the median test, Mann-Whitney Rank U-Score, and Wilcox's T-criterion (in the case of dependent groups) were used for multiple comparison.Statistica for Windows version 8.0 (Stat Soft inc., USA), Microsoft Excel 2007 (Microsoft, USA) software packages were used for statistical and graphical analysis of the obtained results.
Results of the research and their discussion.According to the obtained results (Table ), the intensity of the fibrous reactions in patients with NASH, depending on the presence of a comorbid CKD, indi-cates a probable increase in PBOP blood serum in patients of group 1 by 1.6 times compared with PHP (p < 0.05), patients of group 2 by 2.0 times (p < 0.05), indicating high activity of collagen anabolism in this contingent of patients.At the same time, the index of FOP in blood (Table ), which is the biochemical marker of collagen catabolism, in patients with NASH in group 1 was 1.2 times lower than that in PHP (p < 0.05).That is, in patients with NASH an intensification of collagen formation processes was observed with the background of newly formed collagen resorption processes reduction.At the same time, in patients of group 2, the FOP content in the blood exceeded the data in the PHP by 1.4 times (p < 0.05), indicating an increase in collagen degradation on the background of its high synthesis.The interdependence of the abovementioned changes confirms the presence of a correlation between the content of FOP and α2-MG in blood (r = 0.51, p < 0.05), the content of PBOP and CLA (r = 0.43, p < 0.05); the content of FOP and CLA (r = 0.53, p < 0.05) in group 2.
The analysis of other elements of the extracellular matrix components of protein origin changes in blood, in particular, ceruloplasmin, indicates its probable increase in patients with steatohepatitis of all groups of observation (p < 0,05) with a probable prevalence in patients with NASH in group 2 (1.9 times against 1.4 times in group 1, p < 0.05).We established a strong direct correlation between the values of ceruloplasmin in the blood and the content of bile acids (r = 0.67, p < 0.05), with ceruloplasmin and Alkaline phosphatase activity (r = 0.63, p < 0.05).The increase in the content of osmotic phase proteins that support the quality of inflammation and are activated under conditions of cholestasis, in particular bile acids, is one of the important factors in the progression of fibrosis in the liver.
The analysis of changes in another important component of the protein-derived PCM (Table ) fibronectin belonging to cellular adhesion molecules indicates a probable increase in its content in the blood of patients with NASH with CKD ((1.6 times, p < 0.05), while in patients with NASH its growth was 1.4 times (p < 0.05) compared with the indicator in the PHP.
The established disturbances in the balance of collagen catabolism and anabolism analysis were accompanied by a significant increase in the factors of their regulation of those inductions, in particular, the content of fibroblasts growth factor in the blood (FGF) were more noticed in patients with NASH and CKD (an increase 3.1 times against 2.1 times in Group 1 p < 0.05).These phenomena explains induction phenomenon «Sinusoidal capillary» in patients with NASH with perisinusoidal star cells Ito activation, turning them into myofibroblast-like cells with hyperproduction of collagen in Diss space, the development of pericellular, perissinusoidal, centrolobular and other types of fibrosis on the background of aseptic inflammation around dystrophic (steatosis) of hepatocytes, narrowing of sinusoids and formation of progressive disorders of portal circulation.As the obtained data showed, for the comorbidity of NASH with obesity and CKD, these phenomena were more pronounced and increase faster in comparison with the course of NASH only against the background of obesity.
The obtained data testify that in patients with NASH, which arose on the background of obesity, a significant increase in the synthesis of collagen and glycosaminoglycans was observed.It was accompanied by an ineffective resorption of newly formed collagen due to inhibition of collagenolytic activity of blood plasma at NASH, which arose as a result of activation of proteinase inhibitors (α2-MG), a significant imbalance in the metabolism of CT, which leads to progressive liver fibrosis and violation of its functions.Under conditions of the comorbidity of NASH from the CKD of the I-III stages the collagen synthesis and resorption are activated, but the processes of anabolism predominate, in spite of compensatory activation of collagenolysis, with a significant hyperproduction of actinic-phase proteins, fibronectin, glycosaminoglycans, fibroblast growth factor and increased degradation of extracellular matrix fucoglycoproteins and lead to progressive fibrosis of the liver and disruption of its functions.
Conclusions A significant increase in the synthesis of collagen and glycosaminoglycans was observed in patients with NASH, which was accompanied by an ineffective resorption of newly formed collagen due to inhibition of the collagenolytic activity of plasma, due to significant activation of proteinase inhibitors, a significant imbalance in the system of connective tissue metabolism .Under conditions of the comorbidity of NASH with the CKD of the I-II stages the collagen synthesis and resorption are activated, but the processes of anabolism predominate, in spite of compensatory activation of collagenolysis, with a significant hyperproduction of actinic-phase proteins, fibronectin, glycosaminoglycans, fibroblast growth factor and increased degradation of extracellular matrix fucoglycoproteins and lead to progressive fibrosis of the liver and disruption of its functions.
The authors of this study confirm that the research and publication of the results were not associated with any conflicts regarding commercial or financial relations, relations with organizations and/or individuals who may have been related to the study, and interrelations of coauthors of the article.

Table -
Indicators of the connective tissue components status in patients with non-alcoholic stethogepatitis, obesity and comorbidity with chronic kidney disease