Wnt/ß-catenin signaling in the adrenal glands of rats in various types of experimental hypertension.

Wnt/ß-catenin signaling plays a key role in maintaining homeostasis, which is disturbed in hypertension. Taking into account the lack of literature describing changes in the Wnt/ß-catenin pathway in the adrenal glands under conditions of elevated arterial pressure, here we compare the expression of WNT4, WNT10A, ß-catenin, and GSK-3ß in the adrenal glands of hypertensive rats of various etiologies. The studies were carried out on the adrenal glands of rats with spontaneous hypertension (SHR), renalvascular (2K1C), and deoxycorticosterone acetate (DOCA)-salt. Immunohistochemical and PCR methods were used to identify the molecular components of the canonical signaling pathway and to evaluate gene expression. Immunoreactivity and expression of WNT4, WNT10A, ß-catenin, and GSK-3ß in adrenals of SHR was decreased, compared to control rats. In adrenals of 2K1C rats, intensity of immunohistochemical reaction and expression of WNT4 and ß-catenin was lower, while immunoreactivity and expression of WNT10A and GSK-3ß were higher, compared to normotensive animals. Significantly stronger immunoreaction and expression of WNT4, ß-catenin and GSK-3ß but weaker immunoreactivity and expression of WNT10A were noted in adrenals in DOCA-salt rats, compared to control rats. In conclusion, our data provide new molecular information indicating that the canonical WNT pathway is disrupted in the adrenal glands of hypertensive rats. They show that the dysregulation of the WNT pathway depends on the etiology of hypertension.

Canonical Wnt signaling in the kidney in different hypertension models.

There is a close relationship between the kidney and blood pressure. On the one hand, kidney dysfunction causes an increase in blood pressure; on the other hand, high blood pressure causes kidney dysfunction. Wnt/ß-catenin signaling is a key pathway that regulates various cellular processes and tissue homeostasis and is also involved in damage and repair processes. In healthy organs, Wnt/ß-catenin signaling is muted, but it is activated in pathological states. The purpose of the present study was to immunohistochemically evaluate and compare the expression of WNT4, WNT10A, Fzd8, ß-catenin, and GSK-3ß (glycogen synthase kinase 3ß) in the kidneys of rats with essential arterial hypertension (SHR), renal-renal hypertension (2K1C), and DOCA-salt-induced hypertension. The study was performed on five male WKY rats, seven SHRs, and twenty-four (n = 24) young male Wistar rats. The main results showed that during hypertension, there are changes in Wnt/ß-catenin signaling in the kidneys of rats, and the severity of these changes depends on the type of hypertension. This study is the first to assess the levels of some elements of the canonical Wnt/ß-catenin signal transduction pathway in various types of arterial hypertension by immunohistochemistry and may form the basis for further molecular and functional studies of this pathway in hypertension.

Comparative Assessment of the WNT/ß-Catenin Pathway, CacyBP/SIP, and the Immunoproteasome Subunit LMP7 in Various Histological Types of Renal Cell Carcinoma.

OBJECTIVE: The Wnt/ß-catenin pathway plays an important role in pathogenesis of variety cancers. Most studies on changes in WNT/ß-catenin pathway in renal cell carcinoma (RCC) apply only to clear cell RCC, while there are no comparative assessments of this signaling pathway in various histological types of renal tumors in the available literature. Additionally, considering the close relationship between WNT/ß-catenin signaling, CacyBP/SIP and proteasomal activity, it seemed worth comparing WNT/ß-catenin pathway, CacyBP/SIP and LMP7 immunoproteasome subunit in human samples of clear cell, papillary, and chromophobe RCC. METHODS: Tests were performed on sections of three types of kidney tumors together with surrounding unchanged tissue fragments collected from 50 patients. Samples were divided into three groups depending on the histological type of cancer: clear cell, papillary and chromophobe RCC. Immunohistochemistry and PCR methods were used to identify WNT10A, Fzd5, ß-catenin, GSK-3ß, CacyBP/SIP, LMP7, and gene expression. RESULTS: Immunoreactivity and expression of WNT10A, Fzd5, ß-catenin, GSK-3ß, CacyBP/SIP, LMP7 in clear cell RCC was markedly increased compared to non-cancerous kidney tissue. In papillary RCC, immunoreactivity and expression of WNT/ß-catenin pathway, CacyBP/SIP, LMP7 was also increased compared to non-malignant kidneys, but it was less pronounced than in clear cell RCC. The least substantial increase in immunoreactivity and expression of WNT/ß-catenin pathway, CacyBP/SIP, LMP7 was found in chromophobe RCC, compared to other RCC histological subtypes studied. CONCLUSIONS: Study results suggest an important role of WNT/ß-catenin pathway, CacyBP/SIP and LMP7 in RCC carcinogenesis, and may indicate new aspects of pathomechanisms leading to differences in the biology of clear cell, papillary and chromophobe RCC.

Ageing-related changes in the levels of ß-catenin, CacyBP/SIP, galectin-3 and immunoproteasome subunit LMP7 in the heart of men.

Aging is a major risk factor for morbidity and mortality from cardiovascular causes in men. To better understand the cellular processes related to age-related cardiac complications, we undertook research aimed at comparative evaluation of genes expression and distribution of ß-catenin, CacyBP/SIP, galectin-3 and LMP7 in the heart of healthy men in different age groups. The study was conducted on the hearts of 12 men (organ donors) without a history of cardiovascular disease, who were divided into two age groups: men under and men over 45 years of age. On paraffin sections, immunohistochemical reactions were performed to detect ß-catenin, CacyBP/SIP, galectin-3 and immunoproteasome subunit LMP7. The expression of genes coding ß-catenin, CacyBP/SIP, galectin-3 and LMP7 was also evaluated by real-time PCR method. In the heart of men over 45 years old, both gene expression and immunoreactivity of ß-catenin, CacyBP/SIP, galectin-3 and LMP7 were stronger compared to younger individuals. The results of the presented studies suggest that ß-catenin, CacyBP/SIP, galectin-3 and immunoproteasomes might be involved in the internal regulation of heart homeostasis during ageing.

Mechanism of pro-apoptotic action of prosthetic restorations on oral mucosa cells.

PURPOSE: The aim of this study was to check the pro-apoptotic mechanism of prosthetic reconstruction on epithelial cells of the oral mucosa. PATIENTS AND METHODS: The research was carried out on the saliva of healthy patients using prostheses. The sample swabs were stained using the May-Grünwald-Giemsa method and processed by immunohistochemistry for nuclear factor kappa B (NF-κB; p65) and caspase-3. Western blots were used to detect caspase-3, NF-κB, p53 and COX-2 expression. RESULTS: We found an increased expression of caspase-3, NF-κB and p53 in the oral epithelial cells of patients using prosthetic restorations compared to the subjects from the control group. No differences in COX-2 expression were found between the groups. The strongest immunoreactivity and expression of caspase-3, NF-κB and p53 were observed in patients using full prosthesis for less than two years. CONCLUSIONS: The results of the conducted research indicate that prosthetic restorations may affect the process of apoptosis of oral mucosa epithelial cells. Lack of difference in expression of COX-2 in the saliva of the studied patients suggests that apoptosis is not caused by inflammatory factors.

Differences in leptin biosynthesis in the stomach and in serum leptin level between men and women.

BACKGROUND AND AIM: Although the stomach has been identified as an important source of leptin, the detailed biosynthesis sites of leptin in human gastrointestinal tract have not been fully elucidated. The study objective was to compare leptin distribution and expression in the stomach and its serum level between healthy men and women. METHODS: Nineteen subjects (organ donors; 10 men and 9 women) with normal gastric mucosa histology were recruited. Research material contained gastric samples from the cardia, fundus, and pyloric regions. Gastric mucosa leptin content and leptin gene expression were determined by immunohistochemistry and real-time polymerase chain reaction method. Plasma leptin level was measured using ELISA method. RESULTS: In the stomach of healthy adult subjects, leptin-immunoreactive cells were mainly found in the fundus, and the number of immunoreactive cells was higher in women than in men. Leptin-containing cells were less numerous in the cardia and pylorus mucosa. Similarly, leptin gene expression was the highest in the fundus and higher in women than in men. Serum leptin level was higher in women than in men and was found to correlate positively with body mass index and weight in both sexes. A negative correlation between leptin level and age was noted in women, but not in men. CONCLUSIONS: The current study is the first to provide evidence for the presence of leptin-containing cells in all segments of the human stomach. The differences in gastric leptin biosynthesis and serum leptin levels between men and women suggest that leptin secretion can be controlled by sex hormones or other unknown factors.

Sex differences in distribution of cannabinoid receptors (CB1 and CB2), S100A6 and CacyBP/SIP in human ageing hearts.

BACKGROUND: Women live about 4 years longer due to lower prevalence of cardiovascular complication with ageing. However, the mechanisms involved in the preservation of heart functionality in women have not been fully elucidated. The endocannabinoid system fulfils a significant role in the regulation of cardiovascular system functioning. Cannabinoids, acting through specific receptors (CB1 and CB2), influence on blood pressure, heart rate and myocardial contractility. The function of cardiac muscle cells is strictly dependent on calcium ions. Calcium homeostasis in cardiomyocytes is subjected to complex regulation via calcium-binding proteins. Among them, increasing attention has been paid to the recently discovered S100A6 and CacyBP/SIP. In order to better understand sex differences in the regulation of cardiomyocyte function during ageing, we undertook the present research aimed at immunohistochemical identification and comparative evaluation of cannabinoid receptors, S100A6 and CacyBP/SIP, in the myocardium of ageing men and women. METHODS: The study was conducted on the hearts of 12 men and 10 women (organ donors) without a history of cardiovascular disease. The subjects were divided into two age groups: subjects older than 50 years and subjects under 50 years old. Paraffin heart sections were processed by immunohistochemistry for detection of cannabinoids receptors, S100A6 and CacyBP/SIP. In the heart samples from each study, participant's expression of genes coding for CB1, CB2, S100A6 and CacyBP/SIP using real-time PCR method was measured. RESULTS: CB1 and CB2 immunoreactivity in the cytoplasm of cardiomyocytes in the heart of subjects over 50 was weaker than in younger individuals. In the heart of younger men, CB1-immunoreactivity was weaker and CB2-immunoreaction was stronger compared to women. In the hearts of older men, the CB1-immunostaining was more intense and CB2-immunoreactivity was weaker than in women. Immunodetection of CB1 shoved the presence of receptor in the intercalated discs, but only in the hearts of individuals over the 50 years old. In the hearts of older individuals, stronger immunolabelling was observed for S100A6 and CacyBP/SIP. Male hearts had greater S100A6-immunoreactivity (both age groups) but less CacyBP/SIP immunostaining (individuals over 50 years) compared to the age-matched women. The expression of genes coding CB1, CB2, S100A6 and CacyBP/SIP in the human heart was sex and age-dependent. Observed changes between men and women as well as between subject under and over 50 years were consistent with immunohistochemically stated changes in peptide content. CONCLUSION: Together, the data presented here indicate a close interaction between ageing and sex on the distribution and levels of cannabinoid receptors (CB1, CB2), S100A6 and CacyBP/SIP in the human heart.

Comparative evaluation of cannabinoid receptors, apelin and S100A6 protein in the heart of women of different age groups.

BACKGROUND: Recent studies have shown a significant role of the endocannabinoid system, apelin and S100A6 protein in the regulation of cardiovascular system functioning. The aim of the study was to compare and evaluate the distribution of cannabinoid receptors (CB1 and CB2), apelin and S100A6 protein in the heart of healthy women in different age groups. METHODS: The study was conducted on the hearts of 10 women (organ donors) without a history of cardiovascular disease, who were divided into two age groups: women older than 50 years and women under 50 years of age. Paraffin heart sections were processed by immunohistochemistry for detection of cannabinoids receptors (CB1 and CB2), apelin and S100A6 protein. RESULTS: CB1 and CB2 immunoreactivity in the cytoplasm of cardiomyocytes in the heart of women over 50 was weaker than in younger individuals. There was also strong immunoreactivity of CB1 in intercalated discs (ICDs) of the heart, only in women over 50. The presence of this receptor in this location was not found in women under 50. Apelin- and S100A6-immunoreactivity in the cardiomyocytes was stronger in older women compared to women under 50.The CB1, apelin and S100A6 immunostaining in the endothelium of myocardial vessels was weaker in women over 50 than in younger women, while intensity of CB2- immunoreaction in coronary endothelium was similar in both groups of women. The results of the study indicate the important role of endocannabinoids, apelin, and S100A6 protein in cardiac muscle function. CONCLUSION: This report might contribute to a better understanding of the role of endocannabinoid system, apelin and S100 proteins in heart function as well as shed new light on processes involved in age-related cardiomyopathy.

Renovascular hypertensive decrease immunoreactivity of cells containing chromogranin A and pancreastatin in the pancreas of rats.

Hypertension significantly increases the risk of hyperglycemia in patients. It is known that chromogranin (CgA) and pancreastatin (PST) are involved in regulation of blood pressure and endocrine function of the pancreas. However, still little is known about the physiology of these hormones' secretion in hypertension. The objective of the study was to examine the effects of hypertension on pancreatic islet cells containing CgA and PST in rats. The studies were carried out on the pancreas of rats. After 6 week period of the renal artery clipping procedure, eight 2K1C rats developed stable hypertension. Cells containing CgA and PST were detected using immunohistochemical method. The hypertension significantly decreased the number of pancreatic endocrine cells immunoreactive to CgA and PST antisera. The differences between the hypertensive and normotensive rats concerned not only the number of endocrine cells but also intensity of reactions. In conclusion, the research results indicate that hypertension causes the diminished biosynthesis of CgA and PST in the pancreas of rats and suggests the participation of those peptides in pancreatic disorders occurring in a state of elevated blood pressure.

Influence of doxazosin on biosynthesis of S100A6 and atrial natriuretic factor peptides in the heart of spontaneously hypertensive rats.

Hypertension frequently results in severe complications in cardiovascular system and histopathological changes in the heart. To better understand the cellular processes and signaling pathways responsible for the proper functioning of the heart, we decided to check whether doxazosin affects the density of structures containing S100A6 and atrial natriuretic factor in the heart of spontaneously hypertensive rats. The aim of this study is to find differences in the density of the structures containing S100A6 and atrial natriuretic factor in the heart of spontaneously hypertensive rats treated with doxazosin compared to untreated animals. Fragments of heart were collected from five spontaneously hypertensive rats and five spontaneously hypertensive rats receiving doxazosin for six weeks (dose 0.1 mg per 1 kg of body weight). On the paraffin sections S100A6 and atrial natriuretic factor peptides were localized in the heart using immunohistochemistry. Positive immunohistochemical reaction for S100A6 was observed in atrial and ventricular cardiomyocytes and in the coronary vasculature. In the heart of hypertensive rats treated with doxazosin the S100A6 immunoreactivity was significantly lower compared to untreated animals. Immunodetection of atrial natriuretic factor in the heart of rats confirmed presence of peptide in atrial myocardium. Delicate atrial natriuretic factor-immunoreactivity was observed also in few ventricular cardiomyocytes. The atrial natriuretic factor-immunosignal was significantly weaker in hearts of hypertensive rats receiving doxazosin compared to spontaneously hypertensive rats untreated. Since we found that doxazosin reduces the levels of S100A6 and atrial natriuretic factor peptides in the heart of spontaneously hypertensive rats, it can be assumed that cardiovascular disorders that occur in hypertension may be associated with disturbances of cellular processes and signaling pathways.

Influence of renovascular hypertension on the distribution of vasoactive intestinal peptide in the stomach and heart of rats.

Arterial hypertension is associated with serious dysfunction of the cardiovascular system and digestive system. Given the relevant role of vasoactive intestinal peptide (VIP) in the regulation of digestion process, control of blood pressure and heart rate as well as cardio- and gastro-protective character of the peptide, it appeared worthwhile to undertake the research aimed at immunohistochemical identification and evaluation of VIP-positive structures in the pylorus and heart of hypertensive rats. Up to now, this issue has not been investigated. The experimental model of hypertension in rats according to Goldblatt (two-kidney one clip model of hypertension) was used in the study. The experimental material (pylorus and heart) was collected in the sixth week of the study. VIP-containing structures were evaluated using immunohistochemical and morphometric methods. The analysis of the results showed a significant increase in the number of immunoreactive VIP structures and in the intensity of immunohistochemical staining in the stomach and in the heart of hypertensive rats. Our findings indicate that VIP is an important regulator of cardiovascular and digestive system in physiological and pathological conditions. However, to better understand the exact role of VIP in hypertension further studies need to be carried out.

Alterations of rat stomach endocrine cells under renovascular hypertension.

PURPOSE: The aim of the present study was to perform immunohistochemical and ultrastructural analysis of gastrin-, synaptophysin (SY)- and atrial natriuretic peptide (ANP)-positive cells in the pylorus of "two kidney, one clip" (2K1C) renovascular hypertension model in rats. MATERIAL/METHODS: In order to identify neuroendocrine (NE) cells, immunohistochemical reactions were performed with the use of specific antibodies against gastrin, SY and ANP. Gastric NE cells were also examined using an electron microscope. RESULTS: The present study revealed a twofold increase in the number of gastrin- and SY-positive cells and a significant decrease in the number of ANP-immunoreactive (IR) cells in the pyloric mucosa of 2K1C rats. Test results obtained with an electron microscope confirmed a change in the activity of the stomach endocrine cells of hypertensive rats. CONCLUSIONS: Immunohistochemical and ultrastructural investigations demonstrated the impact of renovascular hypertension on the neuroendocrine system in the rat stomach. The changes in the total number and ultrastructure of DNES cells proved their undeniable role in the modulation of gastric dysfunction, as a consequence of deregulation of homeostasis-maintaining systems.

Dynamics of cocaine- and amphetamine-regulated transcript containing cell changes in the adrenal glands of two kidney, one clip rats.

Taking into consideration the homeostatic disorders resulting from renal hypertension and the essential role of cocaine- and amphetamine-regulated transcript (CART) in maintaining homeostasis by regulating many functions of the body, the question arises as to what extent the renovascular hypertension affects the morphology and dynamics of changes of CART-containing cells in the adrenal glands. The aim of the present study was to examine the distribution, morphology, and dynamics of changes of CART-containing cells in the adrenal glands of "two kidney, one clip" (2K1C) renovascular hypertension model in rats. The studies were carried out on the adrenal glands of rats after 3, 14, 28, 42, and 91 days from the renal artery clipping procedure. To identify neuroendocrine cells, immunohistochemical reaction was performed with the use of a specific antibody against CART. It was revealed that renovascular hypertension causes changes in the endocrine cells containing CART in the adrenal glands of rats. The changes observed in the endocrine cells depend on the time when the rats with experimentally induced hypertension were examined. In the first period of hypertension, the number and immunoreactivity of CART-containing cells were decreased, while from the 28-day test, it significantly increased, as compared to the control rats. CART is relevant to the regulation of homeostasis in the cardiovascular system and seems to be involved in renovascular hypertension. The results of the present work open the possibility of new therapeutic perspectives for the treatment of arterial hypertension, since CART function is involved in their pathophysiology.

Evaluation of density and distribution of CART-immunoreactive structures in gastrointestinal tract of hypertensive rats.

The prevalence of CART (cocaine- and amphetamine-regulated transcript) throughout the organism, multiplicity of functions fulfilled by that peptide, and the collected evidence confirming CART contribution to blood pressure regulation prompted us to undertake the research aiming to identify, localize, and assess changes in CART-immunopositive structures of the gastrointestinal tract (GI tract) of rats with renovascular hypertension. The two-kidney one-clip model of arterial hypertension was used to evaluate the location and density of CART-containing structures in the stomach (cardia, fundus, and pylorus), duodenum, jejunum, ileum, and colon of hypertensive rats. The study was carried out on the GI tract of 20 rats. Ten rats were subjected to the renal artery clipping procedure and after a 6-week period each of them developed stable hypertension. An immunohistochemical localization of CART was performed on paraffin GI tract sections from all the study animals. CART was detected in the extensive population of neurons, particularly within the myenteric plexuses all along the GI tract, and also in neuroendocrine cells, being especially numerous in the stomach and a few in the small intestine. The hypertension significantly increased the density of CART-positive structures in the rat GI tract. The differences between the hypertensive rats and the control animals concerned not only the density of CART-immunoreactive structures but also the staining intensity. As this study provides novel findings, we are planning further molecular examinations to better understand the impact of hypertension on the functioning and activity of CART in the GI tract.

Is reproduction costly? No increase of oxidative damage in breeding bank voles.

According to life-history theory, investment in reproduction is associated with costs, which should appear as decreased survival to the next reproduction or lower future reproductive success. It has been suggested that oxidative stress may be the proximate mechanism of these trade-offs. Despite numerous studies of the defense against reactive oxygen species (ROS) during reproduction, very little is known about the damage caused by ROS to the tissues of wild breeding animals. We measured oxidative damage to lipids and proteins in breeding bank vole (Myodes glareolus) females after rearing one and two litters, and in non-breeding females. We used bank voles from lines selected for high maximum aerobic metabolic rates (which also had high resting metabolic rates and food intake) and non-selected control lines. The oxidative damage was determined in heart, kidneys and skeletal muscles by measuring the concentration of thiobarbituric acid-reactive substances, as markers of lipid peroxidation, and carbonyl groups in proteins, as markers of protein oxidation. Surprisingly, we found that the oxidative damage to lipids in kidneys and muscles was actually lower in breeding than in non-breeding voles, and it did not differ between animals from the selected and control lines. Thus, contrary to our predictions, females that bred suffered lower levels of oxidative stress than those that did not reproduce. Elevated production of antioxidant enzymes and the protective role of sex hormones may explain the results. The results of the present study do not support the hypothesis that oxidative damage to tissues is the proximate mechanism of reproduction costs.