The methylenetetrahydrofolate reductase C677T mutation induces cell‐specific changes in genomic DNA methylation and uracil misincorporation: A possible molecular basis for the site‐specific cancer risk modification

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer although it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one‐carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one‐carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA‐MB‐435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA‐MB‐435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA‐MB‐435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site‐specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation. © 2008 Wiley‐Liss, Inc.     

Major comorbid disease processes associated with increased incidence of acute kidney injury

Acute kidney injury (AKI) is commonly seen amongst critically ill and hospitalized patients. Individuals with certain co-morbid diseases have an increased risk of developing AKI. Thus, recognizing the co-morbidities that predispose patients to AKI is important in AKI prevention and treatment. Some of the most common co-morbid disease processes that increase the risk of AKI are diabetes, cancer, cardiac surgery and human immunodeficiency virus (HIV) acquired immune deficiency syndrome (AIDS). This review article identifies the increased risk of acquiring AKI with given co-morbid diseases. Furthermore, the pathophysiological mechanisms underlying AKI in relation to co-morbid diseases are discussed to understand how the risk of acquiring AKI is increased. This paper reviews the effects of various co-morbid diseases including: Diabetes, cancer, cardiovascular disease and HIV AIDS, which all exhibit a significant increased risk of developing AKI. Amongst these co-morbid diseases, inflammation, the use of nephrotoxic agents, and hypoperfusion to the kidneys have been shown to be major pathological processes that predisposes individuals to AKI. The pathogenesis of kidney injury is complex, however, effective treatment of the co-morbid disease processes may reduce its risk. Therefore, improved management of co-morbid diseases may prevent some of the underlying pathology that contributes to the increased risk of developing AKI.     

Interrelationship between cardiac hypertrophy, heart failure, and chronic kidney disease: endoplasmic reticulum stress as a mediator of pathogenesis

Synthesis of transmembrane and secretory proteins occurs within the endoplasmic reticulum (ER) and is extremely important in the normal functioning of both the heart and kidney. The dysregulation of protein synthesis/processing within the ER causes the accumulation of unfolded proteins, thereby leading to ER stress and the activation of the unfolded protein response. Sarcoplasmic reticulum/ER Ca2+ disequilibrium can lead to cardiac hypertrophy via cytosolic Ca2+ elevation and stimulation of the Ca2+/calmodulin, calcineurin, NF-AT3 pathway. Although cardiac hypertrophy may be initially adaptive, prolonged or severe ER stress resulting from the increased protein synthesis associated with cardiac hypertrophy can lead to apoptosis of cardiac myocytes and result in reduced cardiac output and chronic heart failure. The failing heart has a dramatic effect on renal function because of inadequate perfusion and stimulates the release of many neurohumoral factors that may lead to further ER stress within the heart, including angiotensin II and arginine-vasopressin. Renal failure attributable to proteinuria and uremia also induces ER stress within the kidney, which contributes to the transformation of tubular epithelial cells to a fibroblast-like phenotype, fibrosis, and tubular cell apoptosis, further diminishing renal function. As a consequence, cardiorenal syndrome may develop into a vicious circle with poor prognosis. New therapeutic modalities to alleviate ER stress through stimulation of the cytoprotective components of the unfolded protein response, including GRP78 upregulation and eukaryotic initiation factor 2α phosphorylation, may hold promise to reduce the high morbidity and mortality associated with cardiorenal syndrome.     

Tubulovascular nitric oxide crosstalk: buffering of angiotensin II-induced medullary vasoconstriction

Studies were designed to determine the source of NO responsible for buffering of the angiotensin II (Ang II)-mediated decrease of blood flow in the renal medulla. Intracellular Ca2+ concentration ([Ca2+]i) and NO production ([NO]i) of pericytes and endothelium of the vasa recta were independently measured with the use of fura 2-AM and 4,5-diaminofluorescein diacetate (DAF-2DA), respectively, in microtissue strips of the vascular bundles of the outer medullary vasa recta. Disruption of the endothelium of the vasa recta by perfusion with latex microspheres enabled imaging of the pericytes. Ang II (1 micromol/L) produced an increase of [NO]i of 19+/-6 U in pericytes of the vasa recta when the vessels were adjacent to medullary thick ascending limbs (mTALs). Pericytes of isolated vasa recta without surrounding mTALs showed a rapid peak increase in [Ca2+]i of 248+/-107 nmol/L, with a sustained elevation of 107+/-75 nmol/L, but did not show an increase in [NO]i to either Ang II (1 micromol/L) or the Ca2+ ionophore 4-bromo-A23187 (5 micromol/L). These observations indicated the lack of Ang II and Ca2+-sensitive NO production in pericytes of the vasa recta. In isolated vasa recta with intact endothelium, Ang II reduced [Ca2+]i from 128+/-28 to 62+/-13 nmol/L and failed to increase [NO]i. However, the Ca2+ ionophore did increase [NO]i in the endothelium (47+/-8 U), indicating the presence of Ca2+-sensitive NO production. Significant increases of [NO]i were observed in single isolated mTALs in response to both Ang II (33+/-6 U) and the Ca2+ ionophore (51+/-18 U). We conclude that Ang II increases [Ca2+]i in pericytes of the descending vasa recta as part of its constrictor action and that this vasoconstriction is buffered by the NO from the surrounding tubular elements, such as mTALs.     

Role of endoplasmic reticulum calcium disequilibria in the mechanism of homocysteine-induced ER stress

Our laboratory demonstrated that hyperhomocysteinemia accelerates atherosclerosis in mouse models through ER stress and activation of the unfolded protein response (UPR). In this study, we tested the hypothesis that homocysteine-induced ER stress may arise from ER-Ca(2+) disequilibria. We found that homocysteine-induced cytosolic Ca(2+) transients in T24/83 cells and human aortic smooth muscle cells (HASMCs). These calcium effects occurred at concentrations of homocysteine in the external medium (1-5 mM) that increase intracellular homocysteine in these cell types. Prolonged homocysteine treatment (5 h) at these exogenous concentrations reduced ER-Ca(2+) emptying evoked by thapsigargin. However, these homocysteine-induced effects on ER-Ca(2+) emptying were of a much smaller magnitude than those evoked by A23187 or thapsigargin (ER stressors known to induce ER stress through ER-Ca(2+) depletion). T24/83 cells stably overexpressing the Ca(2+)-binding ER chaperone GRP78 showed diminished cytosolic Ca(2+) transients induced by homocysteine and reduced ER-Ca(2+) emptying evoked by thapsigargin. Prevention of the homocysteine-induced UPR by cycloheximide pretreatment normalized GRP78 expression and ER-Ca(2+) emptying evoked by thapsigargin. These results are inconsistent with a mechanism of ER stress induction by homocysteine through ER-Ca(2+) depletion.     

Apoptosis in the muscular arteries from young spontaneously hypertensive rats.

OBJECTIVE: The purpose of this study was to test the hypothesis that a different incidence of apoptosis occurs in the mesenteric arteries of the spontaneously hypertensive rat (SHR) compared with its normotensive control the Wistar-Kyoto rat (WKY) at 1-2 weeks of age. DESIGN: We examined the incidence of apoptotic cells in the blood vessel wall of muscular arteries from the SHR and WKY at 1-2 weeks of age using two techniques of apoptosis measurement DNA laddering and 3'-OH end labelling. We also measured the volume of the blood vessel wall components and lumen sizes with the confocal microscope to determine whether a differential incidence of apoptosis occurred between the two rat strains. METHODS: We used phenol/chloroform extraction to isolate genomic DNA and assess DNA fragmentation, with gel electrophoresis to determine DNA laddering, and 3'-OH end labelling, where the enzyme terminal deoxynucleotidyl transferase catalyses the addition of fluorescein-conjugated nucleotides to the cut ends of DNA, to detect in situ DNA fragmentation. The volume per unit length of the blood vessel structural components was measured by optical sectioning with the confocal microscope. RESULTS: We found that the SHR had a significantly decreased incidence of cellular apoptosis over WKY. This was true for both the electrophoretic method where SHR had significantly less fragmented DNA (molecular size < 600 bp) than WKY (P= 0.01), and for the microscopic method where SHR had fewer labelled cells in both the adventitia (P= 0.01) and the media (P= 0.0001) layers of large mesenteric arteries. The volumes of the adventitia, media and lumen in the large mesenteric arteries were similar between the two strains at this age. CONCLUSION: These findings suggest that a differential incidence of cellular apoptosis at the age of 1 -2 weeks may be responsible for the larger media volume found in older SHR and thus contributes to the development of hypertension in these animals.