FEATURES OF LIPID, CARBOHYDRATE METABOLISM AND RENAL FUNCTION IN PATIENTS WITH TYPE 1 DIABETES AND DIFFERENT LEVELS OF ALBUMIN IN THE URINE DEPENDING ON THE LEVEL OF CYSTATIN C

UDC: 616.379-008.64:577.112:616.61-07-084 FEATURES OF LIPID, CARBOHYDRATE METABOLISM AND RENAL FUNCTION IN PATIENTS WITH TYPE 1 DIABETES AND DIFFERENT LEVELS OF ALBUMIN IN THE URINE DEPENDING ON THE LEVEL OF CYSTATIN C Kryvoviaz Yu. O.1, Shevchuk N. А.2, Dzevulska І. V.3, Bandurka N. M.1, Shevchuk Yu. G.1 1National Pirogov Memorial Medical University, Vinnytsya (Pyrogov street, 56, Vinnytsya, Ukraine, 21018), 2Vinnytsia Regional Clinical Highly Specialized Endocrinology Center (Michurin street 32, Vinnytsya, Ukraine, 21010), 3Bogomolets National Medical University (Shevchenko boulevard 13, Kyiv, Ukraine, 01601)


Introduction
The formation and progression of renal failure in patients with diabetes is one of the main and unresolved problems of modern health care. The progress made in recent decades in the prevention, diagnosis and treatment of kidney disease has been impressive, reducing the ageadjusted death rate from chronic renal failure (CRF) by about two-thirds in industrialized countries. However, as the most common cause of hospitalization and mortality in patients with diabetes, CRF remains an exception among these encouraging trends [13].
The development of CRF is due to the complex interaction between genetic, neurohormonal, inflammatory and biochemical changes that affect kidney function. In this regard, the growing clinical importance is given to both general biochemical parameters and preclinical early markers of diabetic nephropathy [11].
In modern diagnostics, the control of diabetes mellitus and its compensation is carried out by measuring the level of glucose in the blood, as well as conducting a glucose tolerance test. Glycated hemoglobin is an indicator of compensation of carbohydrate metabolism during the life of erythrocytes. It allows you to detect diabetes in the early stages, which allows you to start treatment in a timely manner [1,3]. Hyperlipidemia is one of the factors in the progression of diabetic nephropathy, because the process of formation of glomerulosclerosis and atherosclerotic plaque on the vessel wall are similar. This is explained by the structural similarity of glomerular mesangiocytes with arterial smooth muscle cells. Lipids filtered into the primary urine can affect the tubules of the renal tubules. Most patients with type 1 diabetes (T1D) have dyslipidemia: increased levels of triglycerides, total cholesterol, low-density lipoprotein and very low-density lipoprotein. At the same time, low levels of high-density lipoproteins are detected [10].
Determination of serum cystatin C levels allows to calculate the glomerular filtration rate. It was noted that the more severe the renal pathology, the worse cystatin C is filtered in the kidneys and the higher its level in the blood. The level of cystatin C increases significantly in the early stages of renal dysfunction. Renal function may be reduced (by more than 50 %) until the level of creatinine only exceeds the upper limit of normal [4].
The levels of the above indicators are considered one of the most important biological constants that indicate the stability of the internal environment of the organism. However, there is a question of expediency of use of cystatin C as a part of multimarker panels for stratification of risk of chronic renal failure, cardiovascular catastrophes, etc.? Wouldn't multiple markers in a set display the same thing as each one? And what will be the contribution of cystatin C in such cases? Based on the results of modern scientific work, we can say that the use of multi-marker panels is based on the fact that different markers reflect the severity of different pathological processes, which in interaction cause a single pathology [7,14,17]. In our work, the answer to these questions will be possible in the course of comparative studies of the values of biochemical parameters in healthy and sick subjects with different levels of albumin and cystatin C.
The aim of the study was to investigate the differences in lipid, carbohydrate metabolism and renal function in T1D patients with different levels of albumin in the urine depending on the level of cystatin C.

Materials and methods
The sample consisted of 78 men and 62 women aged 22-26 years, with T1D, who underwent inpatient treatment in the therapeutic department № 1 and № 2 of the Vinnytsia Regional Highly Specialized Endocrinology Center. The control group consisted of 8 healthy men and 13 healthy women of the same age.
All patients underwent the procedure of determining the level of microalbuminuria by enzyme-linked immunosorbent assay using spectrophotometry (reagents from ORGenTec, Germany). Regulatory values of microalbumin in the set of reagents used -0-25 μg/ml. Venous blood samples were taken from subjects on an empty stomach in the morning (up to 9 hours) after 10-12 hours of fasting.
Blood glucose was determined by enzymatic, amperometric method on a biochemical analyzer Biosen C_Line, manufacturer EKF Diagnostic (Germany). Determination of glucose content was performed using special sensor chips. W hen the sample is applied to the chip sensors, β-D-glucose is converted enzymatically by glucose oxidase into gluconic acid and hydrogen peroxide, which reacts with the electrode. The measurement result was an electric current that is proportional to the glucose concentration. Reference norms of blood glucose 3.3 -5.5 mmol/L. Blood glucose levels were determined on an empty stomach and 2 hours after a meal, and the mean was calculated.
Patients underwent glycated hemoglobin (HbA1c) testing. For this analysis, the method of high performance liquid chromatography on a D 10 analyzer, manufactured by Bio-Rad, was used. Normative values of this indicator <6 %.
International normalized ratio (INR) was determined by converting the prothrombin ratio in INR according to the table. Manual technique. Manufacturer LLC "Genesis". Reference norms up to 1.0.
Biochemical parameters such as total cholesterol and triglycerides were determined photometrically (using enzymes) on a biochemical analyzer using standard kits from Pointe Scientific (USA). Determination of total cholesterol was performed using phenol and peroxidase. The norm for this indicator is less than 5.2 mmol/L. The level of serum triglycerides was determined by the same method but with lipase and peroxidase, the norm of 0.5-1.67 mmol/L.
The level of cystatin C was determined by enzymelinked immunosorbent assay (ELISA) using the kit RD191009100 Human Cystatin C ELISA company BioVendor (Czech Republic). Regulatory values for cystatin C are 0.57-1.12 mg/L for women and 0.6-1.11 mg/L for men.
Statistical data processing was performed in the license package "Statistica 5.5" using non-parametric methods of evaluation of the obtained results.
In patients men with microalbuminuria and cystatin C>0.9, the value of this indicator was lower (p=0.086) compared with women of the same comparison group (6.106±1.493 mmol/L and 7.342±1.722 mmol/L, respectively).
In men and women with microalbuminuria, in whom the level of cystatin C<0.9, a lower (p=0.069 and p=0.097) level of glycated hemoglobin was found compared with subjects of the same sex with microalbuminuria, in whom the level of cystatin C>0.9, (respectively 8.700±1.109 % and 9.978±1.993 %; 9.036±1.586 % and 10.28±2.04 %).
A lower (p<0.01) international normal ratio was found in healthy women (0.954±0.066) compared to sick women with microalbuminuria (0.886±0.078), in whom the level of cystatin C<0.9.
Patients men with normoalbuminuria and cystatin C>0.9 had a lower (p=0.063) value of the international normal ratio compared to women of the same comparison group (0.908±0.093 and 0.978±0.102, respectively).
In men with normoalbuminuria, in whom the level of cystatin C<0.9, a higher (p=0.084) international normal ratio was found compared with those of the same sex with normoalbuminuria, in whom the level of cystatin C>0. A higher (p=0.096) level of cystatin C was found in healthy men (0.681±0.117) compared to healthy women (0.586±0.072).
An important clinical and diagnostic aspect is the assessment of the risk of progression of kidney damage. A positive correlation between serum cystatin C levels and albuminuria is shown. As a marker of GFR for cystatin C has clear advantages over creatinine, the main of which is the ability to recognize the earliest changes in this parameter [8,15]. There is evidence that increased levels of cystatin C can detect kidney damage before microalbuminuria. Thus, in the work of M. Takir et al. (2016) an increase in serum cystatin C was found in the group of patients with decreased GFR and without microalbuminuria [16]. Simultaneously with changes in the level of cystatin C in diabetes, there are changes in the complex of biochemical parameters caused by both the pathological process itself and the resulting metabolic changes in the body. There may be an increase or decrease in the content of substances, increase or decrease in the activity of enzymes, the appearance of metabolites or abnormal forms that do not occur in a healthy person, inadequate response to the load of certain substances [5,12].
For various pathological conditions (except genetically determined) biochemical changes are not strictly specific, and therefore mainly take into account such criteria as "more or less", "longer-faster", "presence-absence" of organspecific indicators, isoenzymes, etc. In fact, certain biochemical parameters are evaluated in comparison with indicators in healthy people, the degree and time of occurrence in the body, changes in the level of an indicator depending on the severity, duration of development and time of manifestation of disorders [2,6,9]. That is why the diagnostic sensitivity of a test is greater, the more adequate its choice and the greater the difference between the indicators in healthy and sick people. Be sure to take into account the differences between men and women, both in normal conditions and in pathology.
In our study in T1D patients, in whom the level of cystatin C<0.9, compared with the control group found: significantly higher values: fasting blood glucose in men with normo-, microalbuminuria and proteinuria -by 30.7 %, 41.1 % and 43.7 %; in women with normo-, microalbuminuria -by 31.9 % and 29.1 %); glucose 2 h after exercise in patients with normo-and microalbuminuria (in men -by 29.3 % and 31.9 %; in women -by 30.9 % and 33.7 %); the average value of glucose in patients with normo-, microalbuminuria and proteinuria -by 26.2 %, 36.5 % and 39.2 % and in women with normo-, microalbuminuria -by 30.9 % and 33.5 %; glycated hemoglobin in sick men with normo-, microalbuminuria and proteinuria -by 45.8 %, 43.4 % and 48.2 % and in women with normo-, microalbuminuria -by 48.5 % and 46.6 %; total cholesterol in sick men with proteinuria by 17.0 %; in women with normoalbuminuria -by 7.6 % and 6.6 %, respectively; triglycerides in sick women with microalbuminuria and proteinuria -by 19.8 %, 49.8 %; cystatin C in patients with normo-and microalbuminuria -by 12.1 % and 9.3 %; and lower values: the international normal ratio in patients with microalbuminuria -by 7.7 %; GFR level by Cockcroft-Gault in sick men with normo-, microalbuminuria and proteinuria -by 17.8 %, 53.7 % and 113.6 %; GFR levels according to CKD EPI in patients with normo-, microalbuminuria and proteinuria -by 10.3 %, 26.8 % and 47.0 %; GFR levels according to cystatin C in patients with normo-, microalbuminuria -by 9.9 % and 9.9 %.
In T1D patients, in whom the level of cystatin C>0.9, compared with the control group found: significantly higher values: fasting blood glucose in men with normo-and microalbuminuria -by 28.0 % and 25.9 %; in women with normo-, microalbuminuria and proteinuriaby 26.4 %, 36.5 % and 46.9 %; glucose 2 hours after exercise in men with normo-and microalbuminuria -by 26.1 % and 30.3 %; in women with normo-, microalbuminuria and proteinuria -by 29.6 %, 31.7 % and 40.2 %; the average value of glucose in men with normo-and microalbuminuria -by 26.9 % and 28.3 %; in women with normo-, microalbuminuria and proteinuria -by 25. 4  With increasing levels of albumin in the urine in patients with cystatin C>0.9 there were changes in the level of the following indicators: increase in patients women with fasting proteinuria by 38.5 %, triglycerides by 74.0 % compared with patients with normoalbuminuria; glycated hemoglobin by 22.5 % and 4.8 %, total cholesterol 33.6 % and 28.9 % compared with sick women with normoalbuminuria and microalbuminuria and a decrease in the international normal ratio in women with microalbuminuria by 7.34 % less compared with women with normoalbuminuria; GFR according to Cockcroft-Gault by 71.8 % compared with women with microalbuminuria and the level of GFR according to CKD EPI by 42.9 % and 43.4 % compared with women with normoalbuminuria and microalbuminuria.
With an increase in the level of cystatin C, a decrease in the level of glycated hemoglobin was found in men by 12.8 % and in women by 12.1 % (microalbuminuria); triglycerides in women by 25.0 % (microalbuminuria) and higher values of the international normal ratio in men by 5.0 % (normoalbuminuria); GFR for cystatin C in men and women with normoalbuminuria and microalbuminuria by 109.0 % and 125.7 %, by 115.6 % and 108.6 %.
Sex differences of the studied indicators are established. Thus, in sick men with cystatin C>0.9 is lower: the international normal ratio by 7.2 %, total cholesterol by 10.8 % (normoalbuminuria); fasting blood glucose by 16.8 % (microalbuminuria) compared with sick women of the same comparison group. In men compared with women of similar comparison groups found: higher level of cystatin C by 62.2 %, Cockcroft-Gault GFR by 23.3 %, CKD EPI GFR by 32.9 % (control group) and lower GRF according to cystatin C by 13.8 % (control group).
Thus, a comparative study of biochemical parameters between healthy and sick subjects with different levels of albumin and cystatin C gives an idea of the functional state of the kidneys, metabolic processes in different organs and the body as a whole, which helps to clarify the nature of the pathological process, pathogenesis and prognosis disease, allows you to judge the effectiveness of treatment.

Conclusions and prospects for further development
1. Between healthy and T1D patients with varying degrees of albuminuria found differences in biochemical parameters, and they (values) are greater the higher the level of cystatin C (except INR, GFR by Cockcroft-Gault, GFR level by CKD EPI, GFR level for cystatin C).
2. Subjects with a cystatin C level <0.9 had lower values of glycated hemoglobin, triglycerides and higher values of the INR, GFR by Cockcroft-Gault and CKD EPI compared with those with a cystatin C level >0.9.
3. In T1D men compared with T1D women found higher values of cystatin C; lower values of INR, total cholesterol (normoalbuminuria) and fasting blood glucose (microalbuminuria).
In further studies it is planned to evaluate the features of lipid, carbohydrate metabolism and renal function in T1D patients depending on the stage of diabetic nephropathy and levels of GRF, which will more accurately interpret the results of research in the clinic.