Pathophysiological and Biochemical Mechanisms of the Development of Diabetic Angiopathies and the Pathogenetic Justification of Their Correction in Experiment

The dissertation is dedicated to the comprehensive study of pathophysiological and biochemical mechanisms of the development of diabetic angiopathies, specifically nephro- and vascular complications in diabetes mellitus. The investigation is conducted using the alloxan diabetes model in rats and includes analysis of changes in macro- and microcirculation, the hemostasis system, and processes of lipid peroxidation and antioxidant defense of the organism. The author pays particular attention to the role of oxidative stress, disturbances of transmembrane ion transport, and the activity of membrane enzymes of renal tissue in the pathogenesis of these diseases.

On the basis of the obtained data, a new methodological approach to correction of identified disturbances using modern antioxidants and membrane-tropic substances is substantiated. The objective of the work is to increase the efficiency of treatment and prevention of vascular complications of diabetes mellitus by optimizing the impact on key pathogenic links at the molecular, cellular, and organ-system levels.

Relevance of the Problem.

Diabetic angio- and nephropathy are not only frequent complications of diabetes mellitus (DM) but also an integral pathogenic link, the frequency and severity of which correlate with the duration of the disease and the degree of metabolic disturbances. Microcirculatory changes are organ-specific in nature and are observed in various regions. Thus, according to Dedov I.I., Suntsov Yu.I., Kudryakova S.V. et al. (1997), among 82.7% of examined DM patients, retinopathy was present in 61.1%, neuropathy in 50.5%, and diabetic nephropathy in 28.2%. In experimental diabetes mellitus, impairment of nerve blood supply and decreased vascular density are noted (Schratzberger P, Walker DH, Ritting K, Bahlmann FH et al; 2001). Diabetic nephropathy is one of the most severe consequences of DM, reducing the quality and duration of life of patients (Dedov I.I., Shestakova M.V., Bondar I.A., Klimentov V.V., Koroleva E.A. et al., 2003) and occupying the first place among other angiopathies. Every second patient with type 1 DM and every fourth patient with type 2 DM dies from acute and chronic renal failure (Suntsov Yu.I., 1998; Dedov I.I., 2003).

In experimental diabetes mellitus, at early stages of its development and in clinical practice in patients, functional disturbances of the fundamental processes — urine formation and renal plasma flow, transepithelial transport of electrolytes, and alterations of the renin-angiotensin-aldosterone system (RAAS), which regulates aldosterone secretion, are observed (Dj Loreto V, Moreno HS, Puche RC et al, 2004; Awad AS, Huang A, Ye H, Duoung ET, Bolton WK, Linden J, Okusa MD, 2005; Pfab T, Thone-Reineke C, Theilig F et al, 2006; Caden MP, Lautenslager OS, HikLE et al, 2007; Hartner A, Cordasic N, Klanke В et al, 2007). Activation of the RAAS and elevation of angiotensin II in the blood are accompanied by renal hypertension and cellular proliferation (Hartner A, Cordasic N, Klanke В et al, 2007). In patients with diabetic nephropathy, in the preclinical stage of development of the pathological process, an increase in systolic blood flow velocity is determined against the background of elevated RAAS activity. Karпов R.S., Koshelskaya O.A., Efimova E.V. et al. (2001) regard impairment of blood flow and its heterogeneity as one of the Dopplerographic signs of initial nephroangiopathy.

In the development of structural-functional abnormalities underlying diabetic angio- and nephropathies and associated pathologies (changes in the blood coagulation system, arterial hypertension), numerous interrelated processes participate. One such disturbance is the imbalance of oxidative-reductive processes and the intensification of uncontrolled free-radical reactions (FRR), termed oxidative stress (Baynes JW, 1991; Tesfamariam В, 1994; Van Damn PC, Van Asbeck BS, Erkelens, 1995).

Systemic metabolic disorders manifest, the character and direction of which allow the assumption that an increase in the intensity of free-radical reactions of membrane phospholipids leads to the accumulation of toxic products of oxidative destruction of lipids, alteration of vascular permeability as a result of endothelial dysfunction, impairment of the functional capacity of membrane structures and the activity of membrane enzymes of cellular metabolism (Kolesnichenko N.S., Mantarova N.S., Shapiro L.A., 1989; Gonskii Ya.I., Korda M.M., Klish I.N., Fira L.S., 1996; Vasilieva O.V., Mobitskii O.B., Klebanova G.K., Vladimirov Yu.A., 1998).

Lipid peroxidation (LP) in platelet membranes contributes to increased functional activity of blood platelets and the release of platelet factors (Kretova E.Yu., Condratev E.I., Sukhanova T.A., 2004; Tokmanova A.Yu., Staroverova D.N., Anisimov M.B., 2004; Remuzzi С et al, 1992; Natsumoto Н, Nakao Т, 2000). The effect of prolonged hyperglycemia on endothelial cells leads to increased production of fibrinolytic factors, more active platelet aggregation, vascular spasm, and damage of capillary endothelium (Volkova A.K., Nedosugova N.I., Rudko I.A. et al., 2000; Kretova E.Yu. et al., 2004; Kawamori R, Jmano Е, Watarai Т et al, 1994; Levin O'Neal's, 2000). These data confirm the presence in patients with diabetic angiopathies of permanent intravascular blood coagulation with participation of the platelet component of hemostasis, which determines the risk of thrombosis development (Bondar T.P., Muratova A.Yu., Pol-Tarabatko N.P., 2004; Khayutina T.L., Balabolkin M.I., Konovalova G.A. et al., 2004).

Taking into account the important role of oxidative stress in the development of diabetic angiopathies, a necessary component of pathogenetic therapy is the use of antioxidants (AO) (Kulagin O.L., 2000; Kokoreva E.V., 2002; Yin X, Zhang Y, Yu J et al, 2006). The study of angioprotective effects of antioxidant preparations in patients with diabetic angiopathies attests to the prospectiveness of this direction (Bobyreva N.V., 1997, 1998; Mashkov Yu.I., 2001; Yan'kova V.I., Ivanova I.L., Fedoreev S.A., Kulesh N.I., 2002; Chugunova N.A., Shamkhala I.Sh., Shestakova M.V., 2004) and to the elevation of antioxidant defense factors and the suppression of lipid peroxidation processes.

Along with well-known antioxidants — α-tocopherol, α-lipoic acid, and others, the literature contains data on the successful use of plant extracts — phytadaptogens (Kulagin O.L. et al., 2001; Yan'kova V.I. et al., 2002). At the same time, there are no investigations dedicated to the study of coupled systems of free-radical reactions and antioxidant defense factors of cells, which underlie disturbances of macro- and microcirculation in peripheral vessels and nephron structures, accompanied by disturbances in the hemostasis system, in experimental diabetes mellitus, as well as their pathogenetic correction.

The relevance of the present research lies in the comprehensive study of pathogenetic mechanisms of development of vascular complications of DM, their correlative relationships at the molecular, cellular, and organ-system levels of organization, which will allow experimental substantiation of new approaches to optimization of treatment and prevention of diabetic angiopathies.

Goal of the Research: to study the pathophysiological and biochemical features of disturbances of interrelated systems of free-radical reactions and antioxidant defense enzymes of cells, which underlie changes in macro- and microcirculation, the hemostasis system, activity of membrane-bound enzymes of renal tissue, and their role in the pathogenesis of diabetic angiopathies. To substantiate new methods of correction of disturbed regulatory mechanisms and to increase the efficiency of treatment and prevention of vascular complications of DM in experiment.

Tasks of the Research:

1. To investigate the nature of changes in macro- and microhemodynamics in rats with experimental diabetes mellitus by Dopplerography.

2. To study the possible role of changes in plasma coagulation and platelet hemostasis in combination with disturbances of tissue perfusion in the development of experimental diabetic angiopathies.

3. To study in experimental diabetes mellitus the disturbances of standard molecular-cellular mechanisms of oxidative stress: membrane mechanisms of transepithelial ion transport, the role of lipid peroxidation (LP) and the antioxidant system of cells (ASC) in the pathogenesis of diabetic nephro- and angiopathies.

4. To investigate correlative relationships between microcirculation, coagulation, and biochemical metabolic disturbances in experimental diabetes mellitus, identifying dependencies that contribute to vascular complications.

5. Using new knowledge of pathophysiological and biochemical mechanisms of pathogenesis of diabetic angiopathies, results of correlation analysis, to substantiate and develop new technologies of prevention and correction using modern antioxidants and membrane-protective substances.

Scientific Novelty. For the first time, a comprehensive approach to the analysis of mechanisms of development of experimental diabetic nephro- and angiopathies using correlation analysis has been employed. New links of pathogenesis have been identified; the participation of disturbances of arachidonic acid metabolism and the antioxidant system in endothelial-dependent regulation of vascular tone, macro- and microhemodynamics, including renal arteries, has been revealed. The role of systemic disturbances of coagulation and platelet hemostasis in the development of diabetic vascular complications has been demonstrated. On the basis of this knowledge, a new methodological approach to correction of identified disturbances has been developed. A positive influence of antioxidants and membrane-tropic substances on the state of renal and general hemodynamics, activity of membrane enzymes of renal tubules, as well as lipid peroxidation and antioxidant defense of cells in experimental diabetes mellitus has been proven.

Scientific and Practical Significance. The obtained results provide new knowledge, deepen and expand the representations of pathogenetic mechanisms of development of vascular complications of diabetes mellitus, substantiate a new, more efficient technology of correction of systemic disturbances using modern antioxidants, membrane-tropic substances, and phytadaptogens.

Practical Value of the Results — in the pathophysiological substantiation of a new methodological approach to prevention and treatment of diabetic angiopathies.

Principal Propositions to be Defended.

1. In experimental diabetes mellitus, disturbances of micro- and macrohemodynamics develop, leading to angiopathies. Tissue perfusion decreases (fluid exchange), mean and systolic blood flow velocities decrease, and diastolic blood flow velocity increases. Changes in hemodynamics in the microcirculatory bed and major vessels (abdominal aorta, renal arteries) are accompanied by disturbances of the coagulation and platelet links of hemostasis and suppression of the fibrinolytic system.

2. Water and electrolyte excretory kidney functions are disturbed as a result of suppression of transmembrane transport of water and electrolytes. The decrease in the level of tubular reabsorption of sodium is due to a decline in Na+,K+-ATPase activity of renal tissue.

3. In experimental diabetes mellitus, the intensity of lipid peroxidation in homogenates of cortical and medullary layers of renal tissue increases, altering the state of the lipid matrix of cell membranes of nephron structures. The antioxidant defense of cells is disturbed.

4. Correction of disturbances of lipid peroxidation and the antioxidant system of cells by antioxidants in combination with membrane-tropic substances leads to a reduction in the degree of expression of diabetic nephropathy, disturbances of hemodynamics, and the hemostasis system.

Dissertation Defense. Materials of the dissertation were reported at: the 61st inter-university scientific conference of young scientists and students "Actual Questions of Modern Medical Science and Healthcare" (Vladikavkaz, 2006); the scientific-practical conference of the Southern Federal District with international participation "Emergency Medicine. Problems of Extreme States" (Vladikavkaz, 2006); the scientific conference of young scientists of SOGMA (Vladikavkaz, 2007); the IV regional scientific-practical conference "New Technologies in Recreation of Population Health" (Vladikavkaz, 2007).

Publications. Based on the materials of the dissertation, 10 works have been published, including in a peer-reviewed journal recommended by VAK RF // "Bulletin of New Medical Technologies", vol.15, №1, 2008, pp.40-41.

Volume and Structure of the Dissertation. The dissertation is presented across 150 pages, including bibliography. It consists of an introduction; a literature review; a description of materials and methods of investigation; four chapters presenting research results, discussion of obtained results, conclusion, general conclusions, and a list of references and appendices. The bibliographic index includes 242 sources of literature, of which 155 are domestic and 87 are foreign authors. The work is illustrated with 15 figures and 13 tables.