RU2634573C1 - Method for stratification of risk of cardiovascular system disorder in patients with chronic kidney disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: level of FGF-23 is determined in the blood plasma of patients with chronic kidney disease by ELISA. An increase in the level of fibroplastic growth factor-23 indicates a high risk of damage to the cardiovascular system.

EFFECT: increased accuracy of cardiovascular disasters risk diagnosis.

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Description

The invention relates to medicine, namely to nephrology, cardiology.

In the blood serum of patients with chronic kidney disease, the level of fibroplastic growth factor-23 is determined by enzyme-linked immunosorbent assay (ELISA). An increase in the level of fibroplastic growth factor-23 indicates a high risk of damage to the cardiovascular system, increases the accuracy of diagnosing the risk of cardiovascular catastrophes in the future.

The increase in mortality of patients on program hemodialysis due to cardiovascular diseases necessitates the search for accessible and reliable criteria for stratifying the risk of developing cardiovascular complications in patients at the pre-dialysis stages of chronic kidney disease. These include: acute cerebrovascular accident, myocardial infarction, exacerbation of chronic heart failure. All cardiovascular catastrophes are based on organic changes in the heart and blood vessels, in particular fibrosis of cardiomyocytes, an increase in their stiffness and obliteration of the arterial wall with a decrease in their lumen. In fact, the main target organs in dialysis patients are myocardium and blood vessels. Moreover, the level of vascular remodeling of the arterial bed is an integral factor determining cardiovascular risks and prognosis for the life of patients with end-stage chronic kidney disease (CKD) [1].

Thus, the search for indicators of the state of the cardiovascular system of patients with early stages of chronic kidney disease is a search for predictors of cardiovascular complications that determine mortality on dialysis.

It is proved that, along with the known factors of the damage to the cardiovascular system, such as arterial hypertension, uremic intoxication, anemia, acid-base disturbance, disorders of bone-mineral metabolism play a crucial role in patients with program hemodialysis. The role of secondary hyperparathyroidism, hyperphosphatemia in the development of renal osteodystrophy and extra skeletal calcification in the development of pathophysiological changes in the cardiovascular system is proved. Later, drugs were created that corrected the levels of not only parathyroid hormone, but also calcium and phosphorus as factors of calciphylaxis. These include calcium mimetics (cinacalcet), phosphate binders and vitamin D. Systolic and diastolic blood pressure, hemoglobin level are subject to active therapeutic correction. However, despite the stabilization of all these indicators, many dialysis patients have organic changes in the myocardium, aorta, and other resistive vessels. Therefore, there is a need to search for those factors whose relief would prevent the remodeling of the cardiovascular system in the conditions of compensation of already known risk factors.

A known method for predicting the outcome of a disease in elderly and senile patients by determining the content of a type 1 matrix matrix metalloproteinase inhibitor in the blood serum of patients. This method involves the identification of the level of tissue inhibitor of matrix matrix metalloproteinases-1 type in blood serum and cardiovascular conjugation, with subsequent determination of the risk of adverse cardiovascular events (application: 2007147240/15, 12/21/2007, authors: Solomakhina N. I., Shchekochikhin D.Yu. RU 2353933 C1).

The disadvantages of this method before the claimed method are:

the failure of a tissue inhibitor of matrix metalloproteinases - type 1, as a marker of vascular remodeling and vascular catastrophes (transient ischemic attacks, ischemic and hemorrhagic stroke, intermittent claudication),

later diagnosis of a high risk of acute myocardial damage, when remodeling processes in the heart are already known, that is, the presence of heart failure is confirmed.

Closest to the claimed method is a method for the diagnosis of heart failure using a quantitative determination of natriuretic propeptides in the blood by ELISA (Russian national recommendations GFCF and OSSN for the diagnosis and treatment of heart failure (second revision), Moscow, 2007). The normal level of the N-terminal inactive fragment of the brain natriuretic peptide (NT-proBNP) in the European population is 0.5-30 pg / ml; the concentration of 125 pg / ml is a threshold, above which the presence of heart failure is predicted. This method is based on the determination of NT-proBNP in plasma by ELISA. Common to the prototype and the proposed method are the following properties:

- determination of the studied markers by ELISA;

- correlation regarding the rapid involvement of the myocardium in the pathophysiological continuum in CKD;

- increase the titer of NT-proBNP and FGF-23 to the appearance of gross organic changes in the myocardium.

Correlation with the degree of systolic dysfunction, the likelihood of exacerbation of heart failure, as well as the degree of risk of death in the next ten years from cardiovascular pathology.

The disadvantages of the prototype, eliminated by the claimed invention, are: the later appearance of a high titer of NT-proBNP in comparison with the claimed method.

The titer NT-proBNP is an indicator that reflects the risk and severity of damage and death of cardiomyocytes, that is, the likelihood of acute heart failure. However, the high level of mNUP in the blood does not reflect the involvement of blood vessels in the pathophysiological continuum and, as a result, the likelihood of a vascular catastrophe (transient ischemic attack, ischemic and hemorrhagic stroke, intermittent claudication).

Great lability of the level of NT-proBNP in the blood even for one hour, since the life span of the most metabolically stable part of the N - terminal fragment of mNUP is 120 minutes, which is significantly less than that of FGF-23. At different periods of the day, the mNUP titer can be of different values. Therefore, this prototype has worse reliability in comparison with the claimed method due to less stability. The essence of the proposed method: the implementation of the invention is achieved as follows. To obtain and prepare material for the study, blood samples of patients in an amount of at least 5 ml are taken. Further, blood samples are clotted for two hours at room temperature or overnight at 4 ° C, then centrifuged for 20 minutes at a speed of 1000 revolutions. For storage, serum samples are aliquoted immediately after receipt and stored frozen at -20 ° C. Samples that will be tested within 5 days can be stored at 2-8 ° C. Before analysis, samples and all components of the kit should slowly reach room temperature.

Standards and samples are added to the appropriate wells of the microplate coated with specific anti-FGF-23 antibodies conjugated to biotin. In the next step, avidin conjugated with horseradish peroxidase (HRP) is added to the wells and incubated for the required time. Then a substrate solution is added to the wells. The change in staining in the wells due to the binding of antibodies conjugated with biotin and avidin conjugated with the enzyme will be observed only in wells containing FGF-23. The enzymatic reaction is stopped by the addition of sulfuric acid, and the intensity of the developed staining is measured using a microplate spectrophotometer at a wavelength of 450 nm ± 10 nm. Next, it is necessary to construct a calibration curve on logarithmic paper by postponing the obtained average optical density (OD) of each standard along the X axis, against the corresponding concentration of each standard along the Y axis, and drawing the optimal curve through the points obtained. For data processing it is recommended to use the appropriate software. The concentration of FGF-23 in the samples is determined by comparing the obtained optical density of the samples with the constructed calibration curve (Fig. 1).

The range of measured concentrations is from 15.6 to 1000 pg / ml. The minimum detectable concentration of FGF-23 is 5.5 pg / ml. Reference values of the normal level of FGF-23 are 10-79 pg / ml. Interpretation of results:

FGF-23 from 80 to 150 pg / ml is a low risk of developing adverse cardiovascular outcomes in the next five years.

FGF-23 150 to 250 pg / ml is a moderate risk of adverse cardiovascular outcomes over the next five years.

FGF-23 from 250 to 500 pg / ml is a high risk of adverse cardiovascular outcomes in the next five years.

FGF-23 from 500 pg / ml or more is an extremely high risk of adverse cardiovascular outcomes over the next five years.

Adverse cardiovascular outcomes are understood as: various variants of coronary heart disease; sudden cardiac death, angina pectoris, chronic heart failure (both systolic and diastolic), acute heart failure (right and left ventricular), acute cerebrovascular accident (both in ischemic and hemorrhagic types), atherosclerosis obliterans of the lower limb arteries.

Damage to the cardiovascular system is the main cause of death for patients on program hemodialysis. Moreover, the frequency of heart damage in chronic renal failure significantly exceeds that in the general population, and the nature of cardiovascular complications is diverse and includes both systolic and diastolic left ventricular myocardial dysfunction (LV), various types of LV hypertrophy, and pathological changes in the vascular wall. A key role in the progression of cardiovascular pathology is played by disorders of bone-mineral metabolism. The pathogenetic continuum of bone-mineral disorders in CKD is complex and multifactorial. The main elements that affect bone metabolism are: hormones eicosanoids - parathyroid hormone, fibroplastic growth factor-23, osteoprotegerin, RANKL, calcitriol, plasma ions - calcium and phosphate. Bone metabolism is controlled mainly by humoral factors and the level of importance of each of them in the genesis of renal osteodystrophy is to this day a cause for discussion. At the same time, the fibroblast-23 growth factor is one of the earliest predictors of bone-mineral and associated cardiovascular disorders.

Fibroblast growth factor-23 (fibroblast growth factor-23, FGF-23) is a phosphatonin protein consisting of 251 amino acids (32 kDa molecular weight) secreted by osteoblasts. The molecular structure is represented by the amino-terminal sequence of the signal peptide (residues 1-24), the central sequence (residues 25-180) and the carboxy-terminal sequence (residues 181-251). FGF-23 exerts its biological effects through the activation of FGF1c receptors. FGF1c receptors, binding to the Clotho cofactor (Engl. Klotho), become 1000 times more sensitive for interaction with FGF-23 than other FGF receptors or Clotho protein alone [2, 4, 5, 6]. Clotho's protein is a 130 kDa transmembrane protein, beta-glucorosonidase. It is expressed mainly in the kidneys and parathyroid glands, the two most important organs involved in the regulation of Ca / P metabolism. It was found that the level of Clotho protein in the body decreases significantly with age. Then its role in the regulation of aging mechanisms was proved. The cloto protein binds to the FGF1c receptor and the C-terminal end of FGF-23, resulting in the conversion of canonical FGF to high affinity specific. Clotho as a co-receptor of FGF-23 is critical for the realization of the biological action of FGF-23, but also has a number of its own effects, independent of it. In kidneys, FGF-23 induces phosphaturia, suppressing the expression of the type IIa and IIc sodium phosphorus cotransporter in the proximal tubules. These are integral proteins on the membranes of tubular epithelial cells. Under the influence of FGF-23, they go inside the cells, which entails a decrease in phosphate reabsorption in the tubules of the nephrons. In addition, FGF-23 suppresses the formation of calcitriol by inhibiting 1-alpha-hydroxylase, which converts 25-hydroxyvitamin D to 1.25 dihydroxyvitamin D and stimulates the formation of 24-hydroxylase, which converts 1.25 dihydroxyvitamin D into inactive metabolites in the proximal tubules of the kidneys . FGF-23 also inhibits the expression of intestinal sodium-phosphorus transporter of intestinal wall epithelial cells, decreasing intestinal phosphorus absorption [4, 5]. The mechanism for lowering the level of phosphorus in the blood is shown in FIG. 2.

The main factor potentiating the release of fibroplastic growth factor-23 is hyperphosphatemia. A clear relationship between the level of FGF-23 and the glomerular filtration rate is determined, since the increase in FGF-23 in the early stages of chronic renal failure is aimed at maintaining a neutral phosphorus balance by increasing the excretion of phosphate in the urine, decreasing gastrointestinal absorption of phosphorus and suppressing calcitriol production. Such a compensatory mechanism is enough to maintain an optimal level of blood phosphate for a long time. However, the progressive loss of renal parenchyma leads to a steadily progressing hyperphosphatemia. And this, in turn, contributes to an increase in the titer of FGF-23. Also, in the sclerosed parenchyma of the kidney, the synthesis of Clotho's co-factor, which is necessary for converting the canonical fibroblast-23 growth factor into high affinity specific and the realization of its physiological effects, is reduced. In patients with advanced stages of CKD, the FGF-23 titer rises tenfold. At the same time, vascular media and myocardium become its point of application. It has been proven that high levels of FGF-23 directly correlate with mortality in dialysis patients, regardless of the level of phosphate or blood pressure. Christian Faul (2000) with a large team of authors showed that a high titer of FGF-23 can directly lead to the development of hypertrophy and myocardial fibrosis. The study consisted of several stages; in the first stage, more than 3000 patients with renal failure were examined, in whom the baseline level of FGF-23 was determined, followed by systematic echocardiographic (EchoCG) control. Left ventricular hypertrophy (LVH) was detected in 52% of patients. Each increase of 1 logarithmic unit FGF-23 (lnFGF23) was associated with an increase in the left ventricular myocardial mass index (LVMI) by 1.5 g / m ² (p <0.001), after correction for other risk factors. Researchers then examined the risk of LVH in 411 patients who had normal echocardiography after 2.9 ± 0.5 years. In 84 patients (20%), LVH was first detected, and in normotensive patients, each increase of 1 unit. lnFGF23 led to an increase in the occurrence of LVH de novo by 4.4 times (p = 0.001), and a high content of FGF-23 caused a 7-fold increase in the frequency of LVH, regardless of the presence or absence of arterial hypertension.

Immunohistochemical and morphometric analyzes of cardiomyocytes under high titer FGF-23 showed a significant increase in their cell surface area, as well as an increase in the level of alpha-actinin protein, indicating an increase in sarcomeres. An increase in the expression of embryonic beta-myosin heavy chains (MTC) and a simultaneous depression of mature alpha-myosin heavy chains were found. Such a switch of ITC isoforms from mature to embryonic indicates a reactivation of the embryonic gene program, which is associated with hypertrophy and remodeling of the heart muscle. FGF-23 also reduces the expression of atrial and cerebral natriuretic peptides (NPs), LVH markers, which can directly lead to diagnostic errors, as the titers of NPs in conditions of excess FGF-23 can be in subnormal values that do not correspond to the real state of the myocardium. FGF-23 reduces the expression of the mid-chain acyl-CoA dehydrogenase (SCAS), an enzyme that regulates the oxidation of fatty acids. Hypertrophic cardiomyocytes switch from energy from fatty acids to carbohydrates, which is a marker of a decrease in the expression of SCAG. However, ischemic processes in the myocardium do not allow the full implementation of both aerobic glycolysis and beta oxidation of fatty acids, which leaves the myocardium without a vital organic substrate. FGF-23 induces LVH irrespective of the Clotho co-receptor, which is expressed primarily in the kidneys and parathyroid glands and is absent in cardiomyocytes, by acting on the FGF4 receptors of cardiomyocytes. The prohypertrophic effect of FGF-23 on cardiomyocytes has been shown. It has also been shown that FGF-23 can act on cardiomyocytes through the FGF4 receptors of cardiomyocytes, regardless of Clotho protein [6, 7, 8].

It is proved that pathophysiological changes in resistive vessels are also associated with the effect of FGF-23 on their muscle layer. As a result, phenotypic transformation of smooth muscle cells that transform into osteoblast-like cells occurs, which entails extra-skeletal mineralization and calcification of the medial layer of arteries. Thus, the elastic properties of the arterial wall are lost [9]. The decrease in the damping function of the arterial system and, first of all, of the aorta leads to the fact that the kinetic energy of the blood flow from the left ventricle to the aorta does not transfer to the potential energy of the expanding aorta, but is realized in increasing the total peripheral vascular resistance. As a result, there is an increase in the afterload on the LV myocardium, which contributes to its hypertrophy, diastolic dysfunction, increased oxygen demand and as a result of its ischemia, fibrosis and systolic dysfunction with a marked decrease in the ejection fraction, the development of severe heart failure. Giorgio Coen et al. (2009) showed an inverse relationship between levels of fetuin A and FGF-23. Meanwhile, it was previously demonstrated that fetuin A synthesized by osteoblasts helps prevent vascular calcification. Thus, the technical result of the claimed invention is to determine the risk of early damage to the cardiovascular system in patients with end-stage CKD. The revealed elevated FGF-23 level from moderate to extremely high untestable numbers indicates, firstly, the high risk of remodeling processes in the cardiovascular system with the initial absence of ECHO-kg signs of myocardial hypertrophy, normal aortic pulse wave velocity, and compensation from other factors risk such as: arterial hypertension, uremia, hyperparathyroidism even in the absence of an increase in such markers of adverse cardiovascular outcomes as high levels of mNUP, endothelin, hyperphosphatemia. Secondly, an increase in FGF-23 indicates the need for cardioprotective therapy, the purpose of which is also the correction of the level of FGF-23 itself. In the absence of drugs that selectively inhibit this mediator, it is proposed to expand the indications for the administration of cinacalcet, which, through the influence on the phosphate-regulating endopeptidase of osteoblasts, reduces the level of fibroplastic growth factor-23. Such early diagnosis of a high risk of long-term organic changes in the cardiovascular system contributes to an early effect on the pathogenetic continuum of CKD and, as a result, to delay or even completely stop the progression of organic changes in the heart and blood vessels, slow the development of LV myocardium hypertrophy with its tonogenic dilatation, and calcification of arteries with a decrease damping function. This, in turn, will increase the life expectancy and improve the prognosis for patients with CKD at all stages and for patients with programmed hemodilysis, which is the long-term goal of therapeutic and diagnostic measures.

List of abbreviations used:

LVH - left ventricular hypertrophy

IHD - coronary heart disease

LVMI - left ventricular myocardial mass index

IFA - enzyme immunoassay

LV - left ventricle

mNUP - cerebral natriuretic peptide

ITC - myosion heavy chains

NUP - natriuretic peptide

OD - optical density

SCAG - middle chain acyl Co-A dehydrogenase

CKD - chronic kidney disease

CRF - chronic renal failure

CHF - chronic heart failure

cGMP - cyclic guanosine monophosphate

ECHO-kg - echocardiography

FGF-23 - fibroplastic growth factor-23

HPR - conjugated horseradish peroxidase

LnFGF23 - FGF-23 Logarithmic Unit

NT-proBNP - N-terminal inactive fragment of brain natriuretic peptide

RANKL - Kappa B Nuclear Factor Activator Receptor

Bibliography

1. Dzgoeva F.U. Osteoprotegerin and the 23rd fibroblast growth factor (FGF-23) in the development of cardiovascular complications in chronic kidney disease / F.U. Dzgoeva, T.M. Gatagonova, T.L. Bestaeva, M.Yu. Sopoev et al. // Therapeutic Archive. - 2014. - No. 6. - S. 63-69.

2. Dzgoeva F.U. Myocardial remodeling in patients with chronic renal failure undergoing treatment with programmed hemodialysis / F.U. Dzgoeva, Z.K. Kadzaeva N.V. Kaloeva et al. // Collection of scientific papers of the North Ossetian branch of the Academy of Sciences of the Higher School of the Russian Federation. Vladikavkaz. - 2006. - No. 4. - S. 253-256.

3. Dobronravov V.A. Dynamics of blood pressure and its daily profile in patients on standard programmed hemodialysis: two-day monitoring data / V.A. Dobronravov // Nephrology. - 2009. - T. 13. - No. 2. - S. 42-49.

4. Dobronravov V.A. A modern view of the pathophysiology of secondary hyperparathyroidism: the role of fibroblast growth factor 23 and Klotho / V.A. Dobronravov // Nephrology. - 2011. - T. 15. - No. 4. - S. 11-20.

5. Nasrallah M.M., El-Shehaby A.R., Salem M.M. et al. Fibroblast growth factor-23 (FGF-23) is independently correlated to aortic calcification in haemodialysis patients // Nephrol Dial Transplant. 2010; 25 (8): 2679-2685.

6. Ix, J.H. Fibroblast growght factor-23 and early decrements in kidney function: the Heart and Soul Study / M.G., Shlipak, C.L. Wassel et al // Nephrol Dial Transplant. - 2010. - No. 25. - P. 993-997.

7. Razzaque M.S. Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process / M.S. Razzaque, D. Sitara, T. Taguchi et al. // FASEB J. - 2006 .-- Vol. 20. - No. 6. - P. 720-722.

8. Wolf M. Forging forward with 10 burning questions on FGF23 in kidney disease / M. Wolf // J Am Soc Nephol. - 2010 .-- Vol. 21. - P. 1427-1435.

9. Gutierrez O.M. Fibroblast growght factor-23 and left ventricular hypertrophy in chronic kidney disease / O.M. Gutierrez, J.L. Janussi, T. Isakova et al. // Circulation. - 2009. - No. 119. - P. 2445-2552.

Claims (1)

A method of stratifying the risk of damage to the cardiovascular system in patients with chronic kidney disease, characterized by the study of blood serum by ELISA, characterized in that the level of FGF-23 is determined and, with a value of more than 80 pg / ml, the risk of damage to the cardiovascular system from low to extremely high, while with a FGF-23 value of from 80 to 150 pg / ml, a low risk is established, from 150 to 250 pg / ml - a moderate risk, from 250 to 500 pg / ml - a high risk, and from 500 or more pg / ml - extremely high risk.

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