Dissertation committee:
Δοντά Ισμήνη, Καθηγήτρια, Ιατρική Σχολή ΕΚΠΑ
Λάζαρης Ανδρέας, Καθηγητής, Ιατρική Σχολή ΕΚΠΑ
Λιάπη Χάρις, Καθηγήτρια, Ιατρική Σχολή ΕΚΠΑ
Τσακίρης Στυλιανός, Αναπλ. Καθηγητής, Ιατρική Σχολή ΕΚΠΑ
Τσαρμπόπουλος Αντώνης, Καθηγητής, Ιατρική Σχολή ΕΚΠΑ
Δάλλα Χριστίνα, Αναπλ. Καθηγήτρια, Ιατρική Σχολή ΕΚΠΑ
Τραφαλής Δημήτρης, Επικ. Καθηγητής, Ιατρική Σχολή ΕΚΠΑ
Summary:
Background and aims:
Choline (Ch) is an essential nutrient involved in a wide variety of metabolic reactions and functions, in both humans and rodents. Besides being a component of various important biological compounds (including the membrane phospholipids lecithin, sphingomyelin and plasmalogen, the neurotransmitter acetylcholine and the platelet activating factor), Ch is involved in many physiological functions (i.e. methyl groups metabolism, lipid transport, second messengers’ generation, etc). Thus, Ch is of paramount importance for the optimal development and function of a number of organs and biological systems among which is the cardiovascular system and the kidneys.
Ch-deprivation has received considerable attention due to its association with adverse health outcomes that can occur across the life span. In 1998, the Food and Nutrition Board of the Institute of Medicine (IOM) and recently, in 2016, the European Food Safety Authority, recognized Ch as an essential nutrient and established dietary intake recommendations. Ch-deprivation can be seen in physiological conditions (e.g. intensive exercise, pregnancy, lactation) and pathological conditions (e.g. alcoholism and malnutrition) and has been associated with heart function impairment, insulin resistance, abnormal fat metabolism and renal failure. Myocardial dysfunction and renal failure also consist two major complications of diabetes mellitus. The term ‘diabetic cadiomyopathy’ has emerged to describe the presence of myocardial dysfunction in the absence of overt clinical coronary artery disease, valvular disease, and other conventional cardiovascular risk factors, such as hypertension and dyslipidaemia; the term diabetic nephropathy refers to chronic loss of kidney function in relation to damage of glomerulai.
Population with diabetes is increasing at alarming rates worldwide and similarly, according to recent studies, the number of people prone to choline deficiency are also constantly increased. It is estimated that only 10% of the US-population is receiving adequate intake of Ch Thus, through multiple pathophysiological mechanisms, the two events (Ch-deprivation and diabetes mellitus) could potentially coexist and interaffect myocardium and renal structural integrity and performance
The aim of the study was to investigate the impact of dietary Ch-deprivation on cardiac and renal function in a streptozotocin (STZ)-experimentally induced diabetic setting in adult male Wistar rats.
Material and Methods:
Twenty-four 3-months-old adult male albino Wistar rats (body weight 298.6 ± 26.2g), were divided into four main groups (n=6 at each group), as follows: Control, diabetic (DM), Ch-deprived (CD), and diabetic Ch-deprived (DM+CD). The induction of diabetes was performed by a single intraperitoneal injection of STZ (50 mg/kg of body weight with dilution in a 0.1 mol/L citrate solution, pH 4.5). The standard diet was enriched by choline (1.5 g/kg) at the expense of sucrose. The analytical composition of choline-deficient diet was the following (g/kg): sugar 413, starch 110, dextrine 110, hydrogenated vegetable oil 100, pea protein 90, soya protein isolate 60, corn oil 50, mineral mix 35, vitamin mix 10, cellulose 10, vitamin-free casein 10, L-cystine 2, in addition to protein ingredient (12%), fat (16%), fiber (2%), ash (3.5%). Rat body weight was assessed weekly and animals were sacrificed after five weeks. Before euthanasia the animals underwent an echocardiography session and upon the animal sacrifice, blood was collected from the aortic arch and used for the estimation of the biochemical parameters (glucose, insulin, homocysteine, lipid profile, urea and creatinine). Hearts and kidneys were used for histological examination along with vascular endothelial growth factor (VEGF-A) and kidney injury molecule -1 (KIM -1) (kidneys only) immunohistochemistry.
Statistical analysis was performed usingGraphPad Prism 5.3.
9.1 Effect of Dietary choline-deprivation on diabetic cardiomyopathy
Results
Measurement of blood glucose levels revealed a successful induction of diabetes in the STZ-treated groups with concurrent hypoinsulinaemia.
After five weeks of dietary intervention, at the echocardiographic assessment diabetic rats under choline deprivation exhibited an increased left ventricular (LV) wall tension index with a concomitant increased of LV velocity and a significant decreased LV posterior wall thickness as compared to diabetic rats fed standard diet. Furthermore, histopathology demonstrated an exacerbation of myocardial inflammation and fibrosis in the diabetic rat myocardium as a result of Ch-deprivation. VEGF immunohistochemistry expression was significantly upregulated in all groups compared to control.
Discussion
In previous studies our group reported the early deleterious effect of Ch-deprivation on the adult healthy rat myocardium. The dietary Ch-deprivation can exacerbate experimentally-induced diabetic cardiomyopathy in rats, within five weeks. The novel findings of this study demonstrate that choline deficient diabetic myocardium presents with a decreased left ventricular wall thickness and higher left ventricular wall tension index along with worsened cardiac inflammation and fibrosis as compared to the diabetic myocardium. The VEGF upregulated myocardial expression reflects the cardiac response to restore its impaired architecture and thus preserve its proper function. The herein presented findings are of particular importance since the examined experimental approach introduces a previously unknown comorbidity simulation of two metabolic disorders that seem to trigger the transition of a restrictive type of cardiomyopathy to a potential dilated type. Further investigation is required to determine the progress of this cardiomyopathy over time.
Moreover, considering the major role of oxidative stress in the development of diabetes-induced complications, as well as previous findings of ours on the cardioprotective role of carnitine in attenuating Ch-deprivation induced cardiotoxicity, the herein presented experimental setting could be the beginning for the further exploring the role of Ch in preventing (or at least modifying) the progress of diabetic cardiomyopathy and the treatment strategies.
9.2 Effect Dietary choline-deprivation effect on diabetic nephropathy
Results
The Ch-deprived diabetic rats exhibited a significant deterioration in renal function with hyperthrophied kidneys (increased kidney weight index) associated with significant alterations in renal architecture characterized by significant increase in renal tubular necrosis, mesengial matrix expansion, and tubulo-interstitial fibrosis. Furthermore, a significant upregulation of the tubular injury marker (KIM-1) was found, while VEGF was significantly downregulated in this design.
Discussion
Dietary Ch-deprivation developed renal lesion resembling to the one induced by ischemia-reperfusion model for renal damage The upregulation of renal KIM-1 (a crucial specific biomarker for diagnosis of early acute kidney injury both in humans and rodents) in choline deprived diabetic rats, in relation to increased blood urea and creatinine levels along with the histopathological findings, confirm the fact that the combined two insults act synergistically to impair renal function and to exacerbate structural damage. Since VEGF dysregulation is considered important factor in triggering the pathogenesis of diabetic nephropathy by inducing glomerulosclerosis and tubular atrophy and subsequent deterioration of renal function the VEGF downregulation in the kidneys of Ch-deprived and Ch-deprived diabetic rats reflects the detrimental effects of Ch deprivation in renal structure and function, It seems that Ch-deprivation acts as a co-factor which complicates the STZ-induced diabetic kidney pathology and the immunohistochemical findings of KIM-1 and VEGF are in line with the renal histopathological picture. This model, diabetes in a choline deprived setting, could probably simulate a clinical case resembling to the one seen in diabetic patients with advanced stage nephropathy.
9.3 Clinical Perspectives
The combined setting of Ch-deprivation and diabetes could eventually resemble common cases in clinical practice, including diabetic patients on parenteral nutrition and/or short bowel syndrome/ and/or autoimmune diseases, diabetic patients with chronic renal failure or hepatic failure and/or alcoholism and/or malnutrition, diabetes in pregnancy/lactating and post-menopausal women, but also critically ill patients.
Diabetic cardiomyopathy and diabetic nephropathy are common serious complication of diabetes mellitus that might appear in the early disease stages and are associated with substantial morbidity and mortality. Taking into account the frequency of the potential inadequate dietary intake of Ch and its interaction with the progress of diabetic cardiomyopathy nephropathy, it turns out that dietary interventions could act as an adjunctive weapon to handle the cardiac dysfunction clinical entity without adding new side effects to the current pharmacological remedies.
