Arnold Kahn

The role of epigenetics in aging and age-related diseases

The role of epigenetics in aging and age-related diseases is a key issue in molecular physiology and medicine because certain epigenetic factors are thought to mediate, at least in part, the relationship between the genome and the environment. An active role for epigenetics in aging must meet two prior conditions: there must be specific epigenetic changes during aging and they must be functionally associated with the aged phenotype. Assuming that specific epigenetic modifications can have a direct functional outcome in aging, it is also essential to establish whether they depend on genetic, environmental or stochastic factors, and if they can be transmitted from one generation to the next. Here we discuss current knowledge about these matters and future directions in the field.

Clonal osteogenic cell lines express myogenic and adipocytic developmental potential

SummaryClonal osteoblastic cell lines were isolated from neonatal rat calvariae and characterized with regard to a number of features associated with authentic osteoblasts. These included elevated alkaline phosphatase activity (relative to fibroblasts), PTH and PGE2-stimulated increases in cAMP, the predominant synthesis of type 1 collagen, and the production of a mineralized matrixin vitro. By these criteria, five clones with osteoblast-like phenotypes were identified (ROB-C8a, C11, C20, C23, and C26) which varied somewhat in shape, levels of alkaline phosphatase activity, and in responsiveness to PTH and PGE2, C11, C20, and C23 responded to both effector substances, whereas C8a only responded to PTH and C26 only responded strongly to PGE2. Upon further examination, two of the clones (C23 and C26) were also found to exhibit significant muscle myotube formation after reaching confluence, and three of the clones (C8a, C11, and C26) showed marked adipocyte differentiation after treatment with dexamethasone. Overall, these data add further supporting documentation to (1) the suspected ontogenetic relationships of osteoblasts to other connective tissue cells, and (2) the concept that osteoblastic cells associated with neonatal rat calvariae are in various stable stages of differentiation and developmental commitment.

Impact of nutritional factors on incident kidney stone formation: A report from the WHI OS

PURPOSE Increased fluid intake, and decreased dietary sodium and animal protein intake are thought to reduce the risk of kidney stones but the role of calcium intake is controversial. We evaluated the relationship between dietary factors and incident kidney stone formation. MATERIALS AND METHODS Secondary analysis was done of 78,293 women from the prospective WHI OS (Womens Health Initiative Observational Study) with no history of nephrolithiasis who completed the validated food frequency questionnaire. Multivariate logistic regression was used to determine demographic and dietary factors, and supplement use independently associated with incident kidney stones. RESULTS Overall 1,952 women (2.5%) reported an incident kidney stone in 573,575 person-years of followup. The risk of incident kidney stones was decreased by 5% to 28% (p = 0.01) with higher dietary calcium intake and by 13% to 31% (p = 0.002) with higher water intake after adjusting for nephrolithiasis risk factors. Conversely higher dietary sodium intake increased the risk of nephrolithiasis by 11% to 61% (p <0.001) after adjustment with the most pronounced effect in women with the highest intake. Higher body mass index independently increased the risk of incident nephrolithiasis (adjusted OR 1.19-2.01, p <0.001). Animal protein intake was not associated with nephrolithiasis on multivariate analysis. CONCLUSIONS This study adds to the growing evidence underscoring the importance of maintaining adequate fluid and dietary calcium intake. Greater dietary calcium intake significantly decreased the risk of incident kidney stones. In contrast, excess sodium intake increased the risk of incident nephrolithiasis, especially in women with the highest intake. Animal protein intake was not independently associated with nephrolithiasis.

Kidney Stones and Subclinical Atherosclerosis in Young Adults: The CARDIA Study

PURPOSE Recent reports suggest that nephrolithiasis and atherosclerosis share a number of risk factors. To our knowledge there has been no previous examination of the relationship between kidney stones and subclinical atherosclerotic disease. We studied the relationship between nephrolithiasis, and carotid wall thickness and carotid stenosis assessed by B-mode ultrasound in the general community using data from the CARDIA study. MATERIALS AND METHODS The CARDIA study is a United States, population based, observational study of 5,115 white and African-American men and women between the ages of 18 and 30 years at recruitment in 1985 to 1986. RESULTS By the year 20 examination 200 (3.9%) CARDIA participants had reported ever having kidney stones. Symptomatic kidney stones were associated with greater carotid wall thickness measured at the year 20 examination, particularly of the internal carotid/bulb region. Using a composite dichotomous end point of carotid stenosis and/or the upper quartile of internal carotid/bulb wall thickness, the association of kidney stones with carotid atherosclerosis was significant (OR 1.6, 95% CI 1.1-2.3, p=0.01), even after adjusting for major atherosclerotic risk factors. CONCLUSIONS The association between a history of kidney stones and subclinical carotid atherosclerosis in young adults adds further support to the notion that nephrolithiasis and atherosclerosis share common systemic risk factors and/or pathophysiology.

Recombinant TGF‐β1 stimulates bone marrow osteoprogenitor cell activity and bone matrix synthesis in osteopenic, old male mice

We have previously hypothesized that the osteopenic changes seen in the skeletons of old male BALB/c mice are due to reductions in the availability and/or synthesis of bone TGF‐β which results in fewer, less osteogenic marrow osteoprogenitor cells (CFU‐f; OPCs) and lower levels of bone formation. Among other things, this hypothesis would predict that introducing exogenous TGF‐β into old mice (growth factor replacement) should stimulate marrow CFU‐f and increase bone formation. In the present study, we have tested this prediction and, indirectly the hypothesis, by injecting human recombinant TGF‐β1, i.p., into both young adult (4 month) and old mice (24 month). The effects of the growth factor on the skeleton were then assessed by measurements of trabecular bone volume, bone formation, fracture healing, and the number, proliferative, apoptotic, and alkaline phosphatase activity of marrow CFU‐f/OPCs. Our data show that the introduction of 0.5 or 5.0 ug/day of TGF‐β1 into old mice for 20 days 1) increases trabecular bone volume, bone formation and the mineral apposition rate, 2) augments fracture healing, 3) increases the number and size of CFU‐f colonies, and 4) increases proliferation and diminishes apoptosis of CFU‐f in primary bone marrow cultures. Importantly, these stimulatory effects of injected growth factor are apparently age‐specific, i.e., they are either not seen in young animals or, if seen, are found at much lower levels. While these observations do not exclude other possible mechanisms for the osteopenia of old mice, they provide further support for the hypothesis that, with age, diminished TGF‐β synthesis or availability results in a reduction in the marrow osteoprogenitor pool and bone formation. The findings also demonstrate that the latter changes can be reversed, at least transiently, by introducing exogenous TGF‐β1. J. Cell. Biochem. 73:379–389, 1999.

Bone loss (osteopenia) in old male mice results from diminished activity and availability of TGF-β

One of the universal characteristics of the long bones and spines of middle‐age and older mammals is a loss in bone mass (osteopenia). In humans, if this bone loss is severe enough, it results in osteoporosis, a skeletal disorder characterized by a markedly increased incidence of fractures with sequelae that may include pain, loss of mobility, and in the event of hip fracture, even death within a relatively few months of injury. An important contributing factor to the development of osteopororsis appears to be a diminution in the number and activity of osteoblasts responsible for synthesizing new bone matrix. The findings in the present and other similar studies suggest that this reduction in osteoblast number and activity is due to an age‐related diminution in the size and osteogenic potential of the bone marrow osteoblast progenitor cell (OPC or CFU‐f) compartment. We previously postulated that these regressive changes in the OPC/CFU‐f compartment occurred in old animals because of a reduction in the amount and/or activity of TGF‐β1, an autocrine growth factor important in the promotion of OPC/CFU‐f proliferation and differentiation. In support of this hypothesis, we now report that (1) the osteogenic capacity of the bone marrow of 24‐month‐old BALB/c mice, as assessed in vivo, is markedly reduced relative to that of 3–4‐month‐old animals, (2) that the matrix of the long bones of old mice contains significantly less TGF‐β than that of young mice, (3) that OPCs/CFU‐fs isolated from old mice produce less TGF‐β in vitro than those recovered from young mice, and (4) that OPCs/CFU‐fs from old mice express significantly more TGF‐β receptor (Types I, II, and III) than those of young animals and that such cells are more responsive in vitro to exogenous recombinant TGF‐β1. We also find that colony number and proliferative activity of OPCs/CFU‐fs of young mice and old mice, respectively, are significantly reduced when incubated in the presence of neutralizing TGF‐β1 antibody. Collectively, these data are consistent with the hypothesis that in old male mice the reduction in the synthesis and, perhaps, availability from the bone matrix of TGF‐β1 contributes to a diminution in the size and development potential of the bone marrow osteoprogenitor pool. J. Cell. Biochem. 70:478–488.

Kidney stones and subclinical atherosclerosis in young adults: Coronary Artery Risk Development in Young Adults (CARDIA) study

PURPOSE Recent reports suggest that nephrolithiasis and atherosclerosis share a number of risk factors. To our knowledge there has been no previous examination of the relationship between kidney stones and subclinical atherosclerotic disease. We studied the relationship between nephrolithiasis, and carotid wall thickness and carotid stenosis assessed by B-mode ultrasound in the general community using data from the CARDIA study. MATERIALS AND METHODS The CARDIA study is a United States, population based, observational study of 5,115 white and African-American men and women between the ages of 18 and 30 years at recruitment in 1985 to 1986. RESULTS By the year 20 examination 200 (3.9%) CARDIA participants had reported ever having kidney stones. Symptomatic kidney stones were associated with greater carotid wall thickness measured at the year 20 examination, particularly of the internal carotid/bulb region. Using a composite dichotomous end point of carotid stenosis and/or the upper quartile of internal carotid/bulb wall thickness, the association of kidney stones with carotid atherosclerosis was significant (OR 1.6, 95% CI 1.1-2.3, p=0.01), even after adjusting for major atherosclerotic risk factors. CONCLUSIONS The association between a history of kidney stones and subclinical carotid atherosclerosis in young adults adds further support to the notion that nephrolithiasis and atherosclerosis share common systemic risk factors and/or pathophysiology.

A Drosophila Model Identifies a Critical Role for Zinc in Mineralization for Kidney Stone Disease

Ectopic calcification is a driving force for a variety of diseases, including kidney stones and atherosclerosis, but initiating factors remain largely unknown. Given its importance in seemingly divergent disease processes, identifying fundamental principal actors for ectopic calcification may have broad translational significance. Here we establish a Drosophila melanogaster model for ectopic calcification by inhibiting xanthine dehydrogenase whose deficiency leads to kidney stones in humans and dogs. Micro X-ray absorption near edge spectroscopy (μXANES) synchrotron analyses revealed high enrichment of zinc in the Drosophila equivalent of kidney stones, which was also observed in human kidney stones and Randall’s plaques (early calcifications seen in human kidneys thought to be the precursor for renal stones). To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation. Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation. Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.

Production and characterization of an antibody against the human bone GLA protein (BGP/osteocalcin) propeptide and its use in immunocytochemistry of bone cells.

We have generated and characterized an antibody that recognizes the C-terminal sequence of the propeptide of human bone GLA protein (BGP/osteocalcin)(amino acid -26 to -1, with +1 being the amino terminus of the mature protein). The range of sensitivity of the antibody, as determined by enzyme-linked immunosorbent assay (ELISA), was 0.5-250 ng/ml. The antibody effectively recognized pro-BGP in cell layer extracts of transformed cells (KT-005), but did not recognize mature, propeptide-less BGP in the medium from the same cultures. Strong labelling was obtained using this antibody in immunoperoxidase staining or immunofluorescence of both transformed and normal human bone cells in vitro. Monensin significantly altered the intracellular pattern of labelling in immunofluorescence studies, indicating that the recognized antigen was associated with the cellular secretory pathway. We also obtained a specific and strong staining of cells in tissue sections of human fetal bone. Antibodies against the mature protein strongly stained the mineralization front, but did not stain cells to any appreciable level. Newly embedded osteocytes were the predominant cell type stained in such material, suggesting that they may represent the major of BGP in the intact tissue. These observations indicate that BGP synthesis is a late event in osteoblastic development and that antibodies generated against the propeptide sequence are a potentially powerful tool in the analysis of bone tumors and evaluation of osteoblastic differentiation.

Dietary Intake of Fiber, Fruit and Vegetables Decreases the Risk of Incident Kidney Stones in Women: A Women's Health Initiative Report

PURPOSE We evaluated the relationship between dietary fiber, fruit and vegetable intake, and the risk of kidney stone formation. MATERIALS AND METHODS Overall 83,922 postmenopausal women from the Womens Health Initiative observational study were included in the analysis and followed prospectively. Cox proportional hazards regression analyses were used to evaluate the associations between total dietary fiber, fruit and vegetable intake, and the risk of incident kidney stone formation, adjusting for nephrolithiasis risk factors (age, race/ethnicity, geographic region, diabetes mellitus, calcium supplementation, hormone therapy use, body mass index and calibrated caloric intake; and dietary water, sodium, animal protein and calcium intake). Women with a history of kidney stones (3,471) were analyzed separately. RESULTS Mean age of the women was 64±7 years, 85% were white and 2,937 (3.5%) experienced a kidney stone in a median followup of 8 years. In women with no history of kidney stones higher total dietary fiber (6% to 26% decreased risk, p <0.001), greater fruit intake (12% to 25% decreased risk, p <0.001) and greater vegetable intake (9% to 22% decreased risk, p=0.002) were associated with a decreased risk of incident kidney stone formation in separate adjusted models. In women with a history of stones there were no significant protective effects of fiber, fruit or vegetable intake on the risk of kidney stone recurrence. CONCLUSIONS Greater dietary intake of fiber, fruits and vegetables was associated with a reduced risk of incident kidney stones in postmenopausal women. The protective effects were independent of other known risk factors for kidney stones. In contrast, there was no reduction in risk in women with a history of stones.