The effect of the experimental chronic hyperglycemia on the kidney and myocardium

Abstract. The number of patients with diabetes increases annually. Modern forecasts predict that diabetes will be the seventh leading cause of death in 2030. Despite many significant advances in the research of diabetes and the use of new modern treatments, the disease is still progressing, and it is necessary to continue to study the effects of diabetes on human systems and organs: kidney and myocardium. 
Methods. A total of 24 rats of reproductive age (6 months old) were involved in this experimental study. Experimental rats were injected with alloxan intraperitoneally once at a dose of 20 mg/100 g on an empty stomach. In addition, they received a 10% glucose solution 24 hours after alloxan injection and a 5% glucose solution during the experiment. We measured glucose level with Accu-Chek Advantage (Boehringer, Germany) after 2, 12, and 24 hours after alloxan injection, and then weekly. The subjects of the investigation were kidney and heart of the experimental (n=12) and control (n=12) animals for correct comparative analysis. 
Results. The average blood glucose level remained at 11 mmol/L ± 2 mmol/L. During the experimental period, the rats' weight gain, dilation of both ventricles and relative renal weight gain were determined. By the histological examination of the myocardium, we revealed polymorphic nuclei, perinuclear cytolysis, fragmentation, wavy-like deformation of cardiomyocytes, stromal and perivascular edema, uneven filling of blood vessels, and local fibrosis. Thinning of fibrous capsule and cortical layer, destruction of nephrons, and hemorrhages were detected in the kidney. 
Conclusions. Our study confirms the robustness of alloxan-induced hyperglycemia in rats. We came to this conclusion because the early changes in the kidneys and heart are explained by the development of microangiopathies, which is a typical feature of the pathogenesis of diabetes. With prolonged exposure to chronic hyperglycemia, structural disorders of vital organs are worsened. This experimental model could be used for conducting comprehensive research aimed to study the mechanisms of diabetes mellitus, the effects of hyperglycemia on organs and tissues, and correct the complications.


Introduction.
Millions of people of all genders and races have diabetes. The World Health Organization informs about to increase the number of such patients aged 20-79 from 108 million in 1980 to almost 463 million in 2019. Approximately half of them do not even suspect the presence of their disease [1,2]. According to the forecasts of the International Diabetes Federation, given the current trends of urbanization and population growth, the number of adults with diabetes in 2025, 2030 and 2045 will be 438, 578 and 700 million, respectively [3,4]. The number of deaths due to diabetes in 2019 is 4.2 million, which is 11.3% of all deaths in the world. By the end of 2019, the total cost of treating diabetes has reached $ 760 billion, which is about 10% of all health care costs. The countries with the highest prices for the treatment of diabetes in 2019 were the USA, China, and Brazil. Analysts attribute this to a large number of overweight residents. Despite the decline in morbidity in some European countries, the increase is 3.4% annually [5][6][7][8][9][10][11].
In 2013 1.04 million patients with diabetes were registered in Ukraine, including 438 000 men and 605 000 women (age 20-79 years) [12,13]. Diabetes prevalence in the European countries is similarly heterogeneous with an age-standardized comparative prevalence ranging from 2.4% in Moldova to 14.9% in Turkey in 2013. Overall, the raw prevalence of diabetes in Europe in 2013 is estimated to be 8.5%, which corresponds to 56 million cases (age 20-79 years) [14].
It is predicted that diabetes will be the seventh leading cause of death in 2030 [1,2]. Some trends in the stabilization of morbidity in developed countries may not be a reason to suspend preventive work, especially against the background of the Covid-19 pandemic, which is now raging around the world. the virus enters the body, the immune system begins to produce cytokines that coordinate cells in the fight against the pathogen. However, some people have an overproduction of cytokines, which leads to a cytokine storm, which attacks both affected and healthy cells. Experiments on mice with induced hyperglycemia and infected with influenza virus revealed excess cytokine production [15,16]. In addition, studies by Chinese physicians have shown that coronavirus-infected people with comorbidities such as diabetes have a more frequent cytokine storm than patients without comorbidities. Therefore, despite many significant advances in the study of diabetes and the use of modern treatments, the disease is still progressing, and it is necessary to continue to study the effects of diabetes on human systems and organs. The present study aimed to investigate the remodeling of experimental rats' kidneys and myocardium under alloxan-induced hyperglycemia.
Materials and Methods. The experimental animals, white inbred matured male rats (n=24), were housed under standard conditions, with proper diet and water ad libitum at the animal facility of Sumy State University, Ukraine.
Animal treatment and all experimental procedures were performed in compliance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The study was approved by the Ethical Committee of Sumy State University, Ukraine.
Alloxan-induced diabetes is one of the widely used model to induce T1DM in experimental animals [17][18][19][20][21]. For this purpose, we used such a chemical substance as alloxan monohydrate. Alloxan is a urea derivative that causes selective necrosis of the β-cells of pancreatic islets [22,23]. The alloxan was re-dissolved in a 0.9% solution of sodium chloride and injected intraperitoneally once at a dose of 20 mg/100 g on an empty stomach [24]. Taking into account that the alloxan causes a toxic effect on the nephron tubules cells [25,26], the experimental animals additionally received a 10% glucose solution 24 hours after alloxan injection and 5% glucose solution during the experiment.
Blood glucose determination. Blood glucose levels were determined using the glucose oxidase method with Accu-Chek Advantage glucometer (Boehringer, Germany) after 2, 12 and 24 hours and then weekly after alloxan administration. Blood samples were taken from the tail vein. The average blood glucose level remained at 11±2 mmol/L.
1. Organometry. We measured the weight of the organs on analytical scales VLR-200-M (Ukraine). Rats' hearts were dissected by Avtandilov, GG [40], and divided into 4 parts: a free wall of the left ventricle (LV), a free wall of the right ventricle (RV), interventricular septum and atria. We weighted the wall of the LV (LVW) and the RV (RVW) with a proportioned mass part of the interventricular septum. We measured the endocardial surface area of the LV (LVSA) and the RV (RVSA) by the method of indirect planimetry and calculated the planimetric index (PI) as the ratio of RVSA to LVSA.
In kidneys, we investigated the absolute weight, the relative weight (g) of organs was calculated by the following formula: Relative weight = absolute lung weight (g) / rat body weight (g), and we measured the thickness of the cortex.
2. Histological examination. Histological samples prepared by the standard method were stained with hematoxylin and eosin, investigated using the light microscope "OLIMPUS BH-2" and photographed with a digital video camera Baumer/optronic Type: CX 05c (Switzerland).
Statistical analysis. The experimental data were processed and analyzed using MS Office 2016 EXCEL (Microsoft Corp., USA) and STATISTICA 13 (TIBCO Software Inc., 2018). The results were expressed as a mean ± SEM. The difference between the groups was determined using the Student's t-test. A probability level (p-value) of less than 0.05 was considered to be statistically significant.
On visual examination, the kidneys of animals affected by hyperglycemia are visually enlarged, their capsule is tense, smooth, pale gray. The relative weight increases by 27,3% (р < 0,05), the absolute weight changes nonsignificantly.
The histological examination of experimental animals' myocardial samples on day 30 of the experiment reveals the presence of polymorphic nuclei of cardiomyocytes. There is a local loss of integrity (fragmentation) and unidirectionality of muscle fibers. The gaps between the cardiomyocyte fibers are dilated due to the development of stromal edema. Significant violations are observed on the part of the vascular component. In some fields of view, vessels are empty. In others, the aggregation of erythrocytes in vessels, capillary hyperemia is visualized (Fig.1). Continued exposure to chronic hyperglycemia causes even more significant changes in the myocardial structure of experimental rats. On day 60 of the investigation, fibers of cardiomyocytes are deformed wavelike. Some cells are unevenly stained; the perinuclear zone is enlightened (cytolysis). Stromal edema progresses, capillaries are full of blood. In some fields of view, areas of the myocardium are replaced by connective tissue with foci of cellular infiltration There is edema around the vessels (Fig. 2). On day 30 of the experiment, the fibrous capsule of the kidney does not have a clear separation from the cortical substance. The amount of subcapsular nephrons increases. Most glomeruli had smooth contours with a distinct cavity between the glomerulus and the capsule. In some areas of the nephron's glomeruli, the capillaries are partially destroyed, so there is hemorrhage within the capsule. No structural changes аre detected in the renal medulla (Fig. 3). On day 60 of the experiment, the fibrous capsule is almost invisible. The thickness of the cortical layer is uneven. There is a tendency to thin it. Hemorrhages are present in the glomeruli of the nephrons. A pyramid of the renal medulla (in a rat`s kidney it is single) loses its shape and clear contours. Many hemorrhages are visible in the renal medulla (Fig. 4). Discussion. Diabetes mellitus (DM) is a multifactorial metabolic disorder, characterized by chronic hyperglycemia leading to significant physiological, biochemical, and histological changes in the internal organs, including kidneys [40,41]. Diabetic nephropathy is the leading cause of end-stage renal disease. The characteristic histologic changes of diabetic nephropathy include thickening of the glomerular and tubular basement membrane, increase in the mesangial matrix, Kimmelstiel-Wilson nodules sometimes combined with microaneurysms, exudative or hyalinosis lesions, capsular drop and afferent and efferent arteriolar hyalinosis [40]. Several factors related to DN include the effect of Український журнал нефрології та діалізу №3 (71) 2021 Оригінальні наукові роботи Original Papers genetic susceptibility, high glucose, polyol pathway activation, renin-angiotensin system activation, reactive oxygen species, activation of the protein kinase C pathway, increase of advanced glycation end-product and glomerular hyperfiltration [42,43]. There are data that alloxan-induced diabetic rat is the most widely in used studying diabetic nephropathy and histological changes in the rat diabetic nephropathy closely resemble the human disease [44]. Pathophysiology of diabetic nephropathy is considered as the result of the interaction between metabolic and haemodynamic factors [45]. There are data that sodium and water retention plays a dominant role in the initiation and maintenance of systemic hypertension in patients with diabetic nephropathy, whereas the contribution of the renin-angiotensin-aldosterone system is smaller [46]. Disorders of urine secretion and urination complicate disorders of homeostasis, especially waterelectrolyte balance (Fig. 5). Water-electrolyte and metabolic imbalance significantly worsen the morpho-functional state of the cardiovascular system [45]. Therefore, to study the structural changes in alloxan-induced hyperglycemia, we chose kidneys and heart.

Metabolic disoders
Hemodynamic disoders diabetic nephropathy disoders of water-electrolite balance cardiovascular disoders On day 30 of the experiment, changes in the studied organs are associated with microcirculatory disorders. In kidneys, the changes are manifested by partial destruction of glomeruli. Such changes in the kidneys' structure develop as a result of microangiopathies with direct damage to the capillary networks of the nephron, as well as damage to the afferent and efferent arterioles which later lead to glomerular hyperfiltration. Damage to the glomerular membrane causes the appearance of protein in the urine. It is no coincidence that microalbuminuria is an early marker of diabetic nephropathy development. Modern research emphasizes that chron-ic hyperglycemia leads to activation of the renin-angiotensin system. Increasing angiotensin II levels lead to efferent arteriolar vasoconstriction and are associated with albuminuria. Another link in the development of efferent arteriola vasoconstriction is endothelin-1 [47][48][49].
Activation of the renin-angiotensin system leads not only to disorders in the kidneys but also to vasoconstriction of blood vessels in the body as a whole. Therefore, on the 30th day of the experiment, morphological disorders are detected in the myocardium as well. Their manifestations are polymorphic nuclei of cardiomyocytes, local loss of integrity and unidirectionality of muscle fibers, stromal edema, and uneven filling of blood vessels.
On day 60 of the study, the above-described changes in kidneys and heart progress. The fibrous capsule and the cortical layer of the kidney become visually thinner. Damage to the nephrons covers not only the glomeruli but also the nephron tubules. Changes in the myocardial structure are worsened by cytolysis, wavelike deformation of cardiomyocytes and local fibrosis. During the experiment, there is an increase in the mass of the heart`s ventricles and an expansion of their cavities. Such changes are explained in our recent study, which found damage to lung tissue under the influence of chronic hyperglycemia [25].
Therefore, disorders occur in both systemic and pulmonary circulation and explain the development of uniform hypertrophy and dilatation of both ventricles during our research. Disorders of water-electrolyte balance due to impaired renal function cause arrhythmias, which are manifested by contractile damage and wavy deformation of cardiomyocyte fibers on the histological preparations of the myocardium.
Conclusions. Our study confirms the robustness of alloxan-induced hyperglycemia in rats. We came to this conclusion because the early changes in the kidneys and heart are explained by the development of microangiopathies, which is a typical feature of the pathogenesis of diabetes. With prolonged exposure to chronic hyperglycemia, structural disorders of vital organs are worsened. This experimental model could be used for conducting comprehensive research aimed to study the mechanisms of diabetes mellitus, the effects of hyperglycemia on organs and tissues, and correct the complications. Authors