Cell Senescence and Its Implications for Nephrology

In 1985, Kaysen and Meyers (1) stated that “the mechanisms and the full biochemical and physiologic consequences of renal senescence remain to be fully elucidated.” The present discussion highlights recent advances in cell biology that have implications for renal senescence. The study of cellular senescence is an emerging field of research with implications for aging, cancer, and chronic disease. It is the study of the limitations on the survival and function of somatic cells. Cells, tissues, organs, and organisms deteriorate over time and deteriorate more rapidly with stress. This deterioration may contribute to normal aging, chronic diseases, the performance of transplanted cells and tissues, repair of injury, and cancer. The cellular and molecular events in this deterioration may present opportunities for understanding and modifying these processes. This article highlights recent developments and their potential relevance in nephrology. This discussion reflects in part a symposium held at the American Society of Nephrology meeting in November 1999. The issue of senescence in renal transplants has been covered elsewhere (2). Given that detailed reviews of renal senescence are available (3,4,5,6,7), we concentrate on basic developments in studies of somatic cell senescence in vitro and then examine their in vivo significance. Definitions We use the term age to mean the time elapsed since birth. The term renal senescence reflects the structural and functional phenotype associated with aged kidneys. Cellular senescence and replicative senescence refer to an in vitro phenotype of cultured somatic cells that have reached their finite limit for replication, a state that may or may not exist for similar cells in vivo. In vitro studies of aged cells, i.e., derived from an old donor, have to be distinguished from senescent cells, which have developed the in vitro senescence phenotype. There is a difference between replicative senescence and terminal differentiation. Replicative senescence is arrived at by cell division. Terminal differentiation is a programmed phenotype, which responds to environmental clues. Whereas terminal differentiation may be a beneficial phenotype, the senescence phenotype in contrast may be a mixed blessing or even detrimental. Molecular Events in Replicative SenescenceIn Vitro The molecular basis of the in vitro cellular senescence phenotype probably differs between cell types and between species, e.g., humans versus mice (8). Hayflick and Moorhead (9) recognized that cultured human somatic cells in vitro displayed a limitation in their number of cycles. This number of cycles was called their Hayflick number. It was lower in cells from older donors and was unaffected by pausing. Thus, human somatic cells have a mechanism for counting the number of times that they have divided, a “mitotic clock.” They stop irreversibly when this cycle number is reached, and they manifest the state of replicative senescence. Telomere Shortening In human cells, shortening of telomeres is critical to replicative senescence. Telomeres are DNA repeats (TTAGGG) at the ends of chromosomes that shorten in dividing normal cells. Telomeres prevent chromosome ends from being confused with DNA breaks and probably have other functions in tethering and sorting chromosomes. The ends of telomeres must be replicated by the enzyme telomerase, a ribonucleoprotein expressed in germline and in immortal cell populations that maintains telomere length constant. In 1973, Olovnikov (10) proposed the telomere theory: namely, that somatic cells were limited because they cannot fully replicate their telomeres (Figure 1). The Hayflick limit was validated by the demonstration that human fibroblasts in culture lack telomerase, shorten their telomeres with each cycle, and develop replicative senescence when telomere length becomes critical. The critical experiment was the demonstration that transfection of telomerase into cultured human cells extends their life span and replication remarkably (11), thus bypassing the Hayflick limit.Figure 1.: Telomere Hypothesis: Telomerase is active in germline cells, maintaining long stable telomeres, but is repressed in most normal somatic cells, resulting in telomere loss in dividing cells. At M1, the Hayflick limit, there is a presumed critical telomere loss in one or perhaps a few chromosomes signaling irreversible cell cycle arrest. This corresponds to the phenotype of replicative senescence. Transformation events may allow somatic cells to bypass M1 without activating telomerase. When chromosomes become critically short on a large number of telomeres, cells are genomically unstable and enter crisis (M2). Rare clones that activate telomerase escape M2, stabilize their genome, and acquire indefinite growth capacity.Telomeric DNA diminishes by approximately 100 bp in dividing normal somatic cells at each cell doubling. The loss of telomeres can trigger the response to DNA breaks, which results in an organized cellular state, the senescence phenotype (M1 in Figure 1). Cells that are driven to continue dividing by abnormal stimuli develop massive genomic instability or crisis (M2). Germline cells and immortal cell populations like most cancer cell lines possess mechanisms, which are either telomerase activation or an alternative mechanism, to preserve their telomere length indefinitely despite cell division, thus protecting their genome. The state of replicative senescence in human skin fibroblasts includes cessation in replication, altered patterns of gene expression, and resistance to apoptosis. Senescent fibroblasts remain viable for many months, with ongoing RNA and protein synthesis. However, senescent cells cannot be stimulated to enter the S phase of the cell cycle by any combination of growth factors or physiologic mitogens. Senescent human fibroblasts show an enlarged and flat morphology and accumulate lipofuscin pigment and senescence associated β galactosidase (SA-β-GAL) activity. The important alterations in gene expression are probably in the genes that control the cell cycle (see below). Other changes in gene expression include increased expression of the genes encoding Alzheimer's β-amyloid precursor protein, certain metalloproteinases, such as MMP-1, and genes whose products contribute to the extracellular matrix, interferon responses, and inflammation. If tissue senescence in vivo is associated with similar changes, it is conceivable that the products of senescent cells could influence not only the cell loss but also the matrix changes and focal inflammation in aged tissues. The key feature of the senescence phenotype in vitro is irreversible cessation of cell cycling. In human cells, telomere shortening is the crucial event that triggers cell cycle arrest. Telomere length for human cells has been compared to the gasoline supply of a car: It is definitely limiting but is certainly not the only mechanism for stopping. Other stresses may also induce some aspects of senescence in human cells and even more so in mouse cells as discussed below. Nevertheless, in human somatic cells in vitro, critical telomere shortening is the trigger for senescence and is responsible for the Hayflick limit. Mechanism of Cell Cycle Arrest in Senescent Cells Senescence-associated growth arrest is mediated by expression of cell cycle inhibitory genes and by downregulation of positive acting cell cycle regulators. At the center of the machinery for cell cycling are the cyclin dependent kinases (CDK), which receive and integrate regulatory signals. CDK are activated in a two-step manner. Step 1 is engagement of the regulators, the cyclins, which cause conformational changes that partially activate the kinase activity. Step 2 is the phosphorylation of a key threonine, leading to full activation. CDK inhibitors (CKI) prevent or reverse activation of CDK (Figure 2). There are two main families of CKI: the INK4 CKI, e.g., p16INK4a, and the CIP/KIP CKI, e.g., p21WAF1/CIP1.Figure 2.: The p53-retinoblastoma (Rb) pathways: p53 is upregulated by DNA damage and/or telomere shortening. p53 is targeted by MDM2 for ubiquination and degradation. This can be prevented by p19ARF, thereby stabilizing p53. The stabilization of p53 allows induction of genes that are important for apoptosis (Bax) and growth arrest (p21WAF1/CIP1). P16INK4a interferes with D-type cyclin binding to the kinases CDK4/6 to decrease their activity and thus progression through G1/S checkpoint. Hypophosphorylated Rb represses E2F-dependent genes and blocks G1/S progression, whereas E2F alone transactivates many genes that are important for mitosis.The protector of the genome, p53, responds to telomere shortening and controls the G1 arrest checkpoint. In response to DNA injury, p53 levels increase by a posttranscriptional mechanism. This results in the transcriptional activation of p21WAF1, a Cip1 CKI, which can mediate G1 arrest (12). Inactivation of p53 is the most common genetic event in human cancer, and p53 deficiency prevents telomere loss from inducing cell cycle arrest (13). The retinoblastoma-susceptibility tumor suppressor protein (Rb) is a major regulator of cell cycling and the critical substrate of the CDK4 and CDK6. Rb negatively controls passage from G1 into S phase by sequestering transcription factors, such as E2F, that are required for the G1/S transition. The ability of Rb to bind transcription factors is abolished by phosphorylation (14). CDK 4 and 6 phosphorylate Rb and thus activate E2F and the cell cycle. Extension of life span beyond the Hayflick limit can be achieved by viral oncoprotein-induced inactivation of p53 and Rb, indicating that these tumor suppressor pathways are critical mediators of the telomere shortening checkpoint response. P16INK4a (INK4 for “inhibitor of CDK4”) causes G1 cell cycle arrest by specific inhibition of CDK4 and CDK6, preventing Rb hyperphosphorylation and S-phase entry (15). P16INK4a is strongly associated with senescence: As mouse or human cells in vitro approach senescence, they express P16INK4a. The gene for p16INK4a is unusual in that it contains an alternative reading frame that allows it to encode both p16INK4a and another factor, p19ARF 16. Telomerase activity alone is insufficient to immortalize some human epithelial cells; inactivation of Rb and p16INK4a is the second crucial step (16,17). Some oncogenes induce senescence in nontransformed human cells via p16INK4a. For example, ras-induced senescence depends on the expression of p16INK4a and p53, and raf can induce senescence in cells that lack p53 function, probably via p16INK4a alone. P53 induction by ras depends on p19ARF. Senescence May Occur by Fundamentally Different Mechanisms in Humans Compared with Mice Both human skin fibroblasts and mouse embryonic fibroblasts can manifest a senescence phenotype after cycling in vitro. However, the senescence state is mediated by telomere shortening in human cells but not in mouse cells (8,18). The senescent state in mouse cells resembles that in human cells at the Hayflick limit, including expression of p16INK4a, but growth arrest in mouse embryo fibroblasts occurs after fewer cycles and without appreciable telomere attrition, probably because accumulated damage from culture conditions (“culture shock”) triggers the p53 response. Thus, “senescence of cultured cells results from two sources of signals, either of which can induce the expression of a common set of inhibitors of the cell division cycle” (18). One set of triggers is extrinsic and stems from stresses from the environment. Culture stresses such as oxidant injury may simulate the stresses of life in vivo over a longer time frame. The second set of triggers is intrinsic and depends on the machinery that monitors the integrity of telomeres (a “mitotic clock”). Humans have a mitotic clock, and mice do not. Differences in the proliferative capacity of cultured mouse and human cells reflect the extent to which they respond to these signaling pathways. Rat cells may be like mouse cells, because they also have very long telomeres. Molecular Changes in Cultured Aged Cells Recent studies demonstrated that fibroblast lines from older humans (aged fibroblasts) displayed mitotic misregulation (19). The investigators took dermal fibroblast cell lines from young, middle-age, and old individuals and from people with Hutchinson-Gilford progeria. Using DNA microarrays of 6000 genes, they identified 61 that were upregulated or downregulated twofold in old and middle-aged versus young. Surprisingly, many regulate G2/M stage of cell cycle, suggesting that aging alters expression of genes involved in cell division. The authors suggested that aging “may occur gradually and in mosaic patterns” (19). Such experiments have limitations in sampling, and by definition select only replicating cells, thus limiting the conclusions (20). Molecular Basis of the Senescence PhenotypeIn Vivo The theories of aging emphasize cumulative damage in postmitotic cells and exhaustion of the finite capacity for replication in cells with mitotic potential. Reactive oxygen species (ROS) that are generated by cellular respiration cause cumulative damage to lipids, proteins, and DNA (21). Genomic instability can in changes in DNA and in loss of Some have that the aging phenotype reflects an genetic but this the of cumulative damage and finite of the of aging that most of aging is driven by environmental factors aging is by a loss of cells, of programmed cell could a in loss of cells could reflect a finite ability to cells in some cell senescence in vitro causes loss of their ability to apoptosis as as other abnormal suggesting that of senescent cells could contribute to aging and of through factors could contribute to aging, e.g., through loss of of aging can be in vivo. include the of products and a probably reflects of and also occurs in senescent cells in vitro. 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