Glenda C. Gobe

Anatomical methods in cell death

Publisher Summary This chapter describes methods for studying the morphology of cell death and the criteria used in identifying apoptosis and necrosis. Electron microscopy provides the most reliable method for recognizing the two processes; in many cases, however, they can be identified confidently using light microscopy alone. The recognition of apoptosis and necrosis is based primarily on the distinctive changes that take place within the affected cells. However, when these two processes occur in vivo, they also differ in their distribution and in the tissue reactions that are associated with them. These latter features may be of subsidiary use in identification. Thus, apoptosis involves scattered individual cells in a tissue, whereas necrosis involves groups of adjoining cells. Necrosis is accompanied by an acute inflammatory response with exudation of neutrophil leukocytes and monocytes; this event is characteristically absent in apoptosis. The light microscopic recognition of apoptosis depends on the detection of discrete well-preserved apoptotic bodies. Although convoluted budding cells are sometimes observed in smears, they are rarely seen in paraffin sections of immersion-fixed tissue.

Internucleosomal DNA Cleavage Should not be the Sole Criterion for Identifying Apoptosis

Evidence is presented that questions the validity of using DNA electrophoresis in isolation for identifying apoptosis.

Cell Death Induced in a Murine Mastocytoma by 42–47°C Heating in Vitro: Evidence that the Form of Death Changes from Apoptosis to Necrosis Above a Critical Heat Load

The pathogenesis of heat-induced cell death is controversial. Categorizing the death occurring after various heat loads as either apoptosis or necrosis might help to elucidate this problem, since it has been shown that these two processes differ in their mode of initiation as well as in their morphological and biochemical features. Log-phase cultures of mastocytoma P-815 x 2.1 were heated at temperatures ranging from 42 to 47 degrees C for 30 min. After 42 degrees C heating a slight increase in apoptosis was observed morphologically. However, after heating at 43, 43.5 and 44 degrees C, there was marked enhancement of apoptosis, and electrophoresis of DNA showed characteristic internucleosomal cleavage. With heating at 45 degrees C both apoptosis and necrosis were enhanced, whereas at 46 and 47 degrees C only necrosis was produced. DNA extracted from the 46 and 47 degrees C cultures showed virtually no degradation, which contrasts with the random DNA breakdown observed in necrosis produced by other types of injury; lysosomal enzymes released during heat-induced necrosis may be inactivated at the higher temperatures. It is suggested that apoptosis following heating may be triggered either by a limited increase in cytosolic calcium levels resulting from mild membrane changes or by DNA damage. Necrosis, on the other hand, is likely to be a consequence of severe membrane disruption.

Biomarkers in chronic kidney disease: a review

Chronic kidney disease (CKD) is a major public health problem. The classification of CKD by KDOQI and KDIGO and the routine eGFR reporting have resulted in increased identification of CKD. It is important to be able to identify those at high risk of CKD progression and its associated cardiovascular disease (CVD). Proteinuria is the most sensitive marker of CKD progression in clinical practice, especially when combined with eGFR, but these have limitations. Hence, early, more sensitive, biomarkers are required. Recently, promising biomarkers have been identified for CKD progression and its associated CVD morbidity and mortality. These may be more sensitive biomarkers of kidney function, the underlying pathophysiological processes, and/or cardiovascular risk. Although there are some common pathways to CKD progression, there are many primary causes, each with its own specific pathophysiological mechanism. Hence, a panel measuring multiple biomarkers including disease-specific biomarkers may be required. Large, longitudinal observational studies are needed to validate candidate biomarkers in a broad range of populations prior to implementation into routine CKD management. Recent renal biomarkers discovered include neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, and liver-type fatty acid-binding protein. Although none are ready for use in clinical practice, it is timely to review the role of such biomarkers in predicting CKD progression and/or CVD risk in CKD.

Oxidative stress, anti‐oxidant therapies and chronic kidney disease

Chronic kidney disease (CKD) is a common and serious problem that adversely affects human health, limits longevity and increases costs to health‐care systems worldwide. Its increasing incidence cannot be fully explained by traditional risk factors. Oxidative stress is prevalent in CKD patients and is considered to be an important pathogenic mechanism. Oxidative stress develops from an imbalance between free radical production often increased through dysfunctional mitochondria formed with increasing age, type 2 diabetes mellitus, inflammation, and reduced anti‐oxidant defences. Perturbations in cellular oxidant handling influence downstream cellular signalling and, in the kidney, promote renal cell apoptosis and senescence, decreased regenerative ability of cells, and fibrosis. These factors have a stochastic deleterious effect on kidney function. The majority of studies investigating anti‐oxidant treatments in CKD patients show a reduction in oxidative stress and many show improved renal function. Despite heterogeneity in the oxidative stress levels in the CKD population, there has been little effort to measure patient oxidative stress levels before the use of any anti‐oxidants therapies to optimize outcome. This review describes the development of oxidative stress, how it can be measured, the involvement of mitochondrial dysfunction and the molecular pathways that are altered, the role of oxidative stress in CKD pathogenesis and an update on the amelioration of CKD using anti‐oxidant therapies.

High-carbohydrate high-fat diet–induced metabolic syndrome and cardiovascular remodeling in rats.

The prevalence of metabolic syndrome including central obesity, insulin resistance, impaired glucose tolerance, hypertension, and dyslipidemia is increasing. Development of adequate therapy for metabolic syndrome requires an animal model that mimics the human disease state. Therefore, we have characterized the metabolic, cardiovascular, hepatic, renal, and pancreatic changes in male Wistar rats (8-9 weeks old) fed on a high-carbohydrate, high-fat diet including condensed milk (39.5%), beef tallow (20%), and fructose (17.5%) together with 25% fructose in drinking water; control rats were fed a cornstarch diet. During 16 weeks on this diet, rats showed progressive increases in body weight, energy intake, abdominal fat deposition, and abdominal circumference along with impaired glucose tolerance, dyslipidemia, hyperinsulinemia, and increased plasma leptin and malondialdehyde concentrations. Cardiovascular signs included increased systolic blood pressure and endothelial dysfunction together with inflammation, fibrosis, hypertrophy, increased stiffness, and delayed repolarization in the left ventricle of the heart. The liver showed increased wet weight, fat deposition, inflammation, and fibrosis with increased plasma activity of liver enzymes. The kidneys showed inflammation and fibrosis, whereas the pancreas showed increased islet size. In comparison with other models of diabetes and obesity, this diet-induced model more closely mimics the changes observed in human metabolic syndrome.

Molecular cell biology of androgen receptor signalling.

The classical action of androgen receptor (AR) is to regulate gene transcriptional processes via AR nuclear translocation, response element binding and recruitment of, or crosstalk with, transcription factors. AR also utilises non-classical, non-genomic mechanisms of signal transduction. These precede gene transcription or protein synthesis, and involve steroid-induced modulation of cytoplasmic or cell membrane-bound regulatory proteins. Despite many decades of investigation, the role of AR in gene regulation of cells and tissues remains only partially characterised. AR exerts most of its effects in sex hormone-dependent tissues of the body, but the receptor is also expressed in many tissues not previously thought to be androgen sensitive. Thus it is likely that a complex, more over-arching, role for AR exists. Each AR domain co-ordinates a multitude of individual and vital roles via a diverse array of interacting partner molecules that are necessary for cellular and tissue development and maintenance. Aberrant AR activity, promoted by mutations or binding partner misregulation, can present as many clinical manifestations including androgen insensitivity syndrome and prostate cancer. In the case of malignant prostate cancer, treatment generally revolves around androgen deprivation therapies designed to interfere with AR action and the androgen signalling axis. Androgen therapies for prostate cancer often fail, highlighting a real need for increased research into AR function.

The Role of Apoptosis in the Response of Cells and Tumours to Mild Hyperthermia

There is now abundant evidence that apoptosis, the cell death mechanism responsible for physiological deletion of cells, can be triggered by mild hyperthermia. However, the overall importance of this mode of death in heated tumours has not yet been established. In this light and electron microscopic study, apoptosis induced by 43 degrees C or 44 degrees C water bath heating for 30 min in a range of murine and human tumours growing in vitro and in four murine tumours growing as solid nodules in vivo, was identified on the basis of its characteristic morphology, and the amount present quantified. Apoptosis was found to play a variable role in the response of tumours to heating, with the lowest levels produced in human melanoma lines (less than 1%) and the highest levels in some Burkitts lymphoma lines (up to 97%). In these latter tumours the induction of apoptosis is clearly a major component of the hyperthermic response.

Associations of Glomerular Number and Birth Weight With Clinicopathological Features of African Americans and Whites

BACKGROUND Hypertension and its cardiovascular complications affect African Americans more severely than whites, a disparity variously ascribed to low birth weight, low glomerular number, an exaggerated arteriolonephrosclerotic blood pressure response, and inflammation-induced oxidative stress. STUDY DESIGN Case series. SETTING AND PARTICIPANTS Autopsy kidneys of 107 African Americans and 87 whites aged 18 to 65 years at a single medical center between 1998 and 2005. Excluded were persons with known premorbid kidney disease; pathological findings of severe arterioarteriolonephrosclerosis, nodular and diffuse diabetic glomerulosclerosis, or nonischemic cardiomyopathy. PREDICTORS & OUTCOMES Associations of: (1) race, age, sex, birth weight, obesity, and glomerular number (predictors) with hypertension and death from coronary artery (CAD) and cerebrovascular disease (CVD; outcomes); and (2) age, blood pressure, and race (predictors) with arteriolonephrosclerotic changes, including chronic tubulointerstitial inflammation (outcomes). MEASUREMENTS Hypertension ascertained from chart review and heart weight. Cause of death determined from chart review and autopsy findings. Birth weight obtained from birth records (115 persons). Total glomerular number (N(glom)) estimated by using the dissector/fractionator technique. Arteriolosclerosis, glomerulosclerosis, cortical fibrosis, and chronic inflammation by using CD68 density were measured morphometrically. RESULTS 59 African Americans (55%) and 32 whites (37%) were classified as hypertensive. CAD and CVD were the cause of death in 64 (33%) and 18 persons (9%), respectively. By using multiple linear regression, birth weight (P < 0.001) and sex (P < 0.01), but not race (P = 0.3) or age (P = 0.2), predicted N(glom) (P < 0.001; adjusted r(2) = 0.176). Hypertension was associated with African American race (P = 0.04), older age (P < 0.001), and male sex (P = 0.01), but not with N(glom) (P = 0.9), body mass index (P = 0.9), or birth weight (P = 0.4). Hypertension was the only significant factor associated with CAD and CVD (P < 0.001 for both). Interactions of age and blood pressure with race showed that although African Americans had more severe hypertension (P < 0.001) and arteriolosclerosis (P = 0.01) at a younger age than whites, there were no significant racial differences in degrees of arteriolosclerosis, glomerulosclerosis, cortical fibrosis, or CD68 density for any level of increased blood pressure. LIMITATIONS The study is observational and descriptive. CONCLUSIONS The more severe hypertension found in African Americans could not be attributed to racial differences in N(glom) or birth weight. CAD and CVD death and increased arteriolonephrosclerosis, including CD68 density, were determined by using blood pressure without a significant interacting contribution from race.

Apoptosis and expression of Bcl-2, Bcl-XL, and Bax in renal cell carcinomas.

There are at present disparate published results with regard to the relevance of the Bcl-2 gene family, levels of apoptosis, and cell proliferation in the development and progression of renal cell carcinoma (RCC). The present study analyses the inter-relationship between the expression of representatives of the anti-apoptotic (Bcl-2, Bcl-XL) or pro-apoptotic (Bax) Bcl-2 proteins, incidence of apoptosis, and mitosis in a selected small group of 22 graded RCCs that had paired normal renal tissue, or non-neoplastic tissue in the renal biopsy specimen. The cases were chosen to determine the feasibility of measuring these parameters as potential surrogate markers of progression or treatment failure of the cancers. The results showed that in approximately 50% of the RCCs, where Bcl-2 and/or Bcl-XL expression was high, apoptosis was not detected, and when expression of these proteins was low or not found, increased levels of apoptosis were seen. In most of the remaining 50% of samples, high levels of Bcl-XL but not Bcl-2 were negatively correlated with low levels of apoptosis (Bcl-XL: r=−0.437, P=0.07 and Bcl-2: r=+0.560, P=0.02). For the same group of samples, high Bax expression was found in association with apoptosis (r=+0.578, P=0.02). A novel finding was an association between low expression of Bcl-2 and/or Bcl-XL in normal tissue and the level of expression of these proteins in the RCCs, an intrinsic variation that may be an individual patient factor. The results indicate that, in RCCs with increased expression of Bcl-2 and/or Bcl-XL, levels of apoptosis are minimal and these combined factors may assist in progression of the cancers and resistance to treatments.