Aging of the Kidney 289
Cellular Basis of Tissue Aging 291
Molecular Mechanisms Underlying Cellular Aging 292
Lessons from Lower Organisms 292
NAD + -consuming Reactions and Mammalian Cell Health 292
Sirtuins and Cellular Metabolism 293
Sirtuins and Endothelial Cells 293
Sirtuins and Vascular Smooth Muscle Cells 294
NAD + Biosynthesis and Cell Health 295
NAD + -producing Enzymes 295
Nampt is an NAD + -regenerating Enzyme 295
Nampt in Vascular Cell Aging and Regenerative Capacity 295
Aging is an inevitable process that affects renal performance, promotes renal disease and drives risk factors for renal disease. Not surprisingly, therefore, is aging also a major barrier to organ regeneration. Understanding the molecular basis of renal aging, and potentially intervening with the process, will thus be important for realizing the potential of regenerative nephrology. In this chapter, cellular and molecular features of aging, including the phenomena of telomere-dependent and telomere-independent cellular senescence, are discussed. The renal vasculature is particularly susceptible to premature aging, and slowing endothelial cell, smooth muscle cell and pericyte aging could favorably impact renal homeostasis during aging and regeneration capacity. Recently, NAD + production and consumption pathways, including the sirtuin SIRT1-dependent cascades, have emerged as exciting determinants of the balance between regeneration and aging. A better understanding of these cascades could lead to strategies to suppress replicative or stress-induced aging and optimize regenerative efforts.
It is well accepted that preventing the insults that can lead to renal damage is preferable to managing renal disease once it has manifest. This is a feasible strategy in some instances, avoidance of high blood pressure being a prime example. However, as with many conditions, preventing renal damage often is not an option because the pathology has advanced beyond the point at which such a strategy is relevant. In this context, the notion of tissue regeneration is emerging an attractive potential therapy.
Although considered as a therapeutic strategy, regeneration is also an innate property of adult organs, albeit often of limited capacity. Indeed, strategies that harness the innate regenerative capacity of organs may prove to be an effective mode of regenerative medicine. However, the innate response to severe or sustained kidney damage is primarily fibrosis, not regeneration. This reality highlights a challenge to the field of regenerative nephrology, as the barriers to innate renal regeneration will also be barriers to therapeutic regenerative strategies.
In this chapter, one of the major barriers to renal regeneration, namely cell and tissue aging, is discussed. Aging of the vasculature, a process that can profoundly limit functional repair and regeneration, will be emphasized. Molecular pathways that regulate cellular aging will be reviewed, focusing on the production and consumption pathways of the oxidized form of nicotinamide adenine dinucleotide (NAD + ), including sirtuin-1 (SIRT1)-dependent cascades, cascades that are emerging as key determinants of regeneration versus aging.
Aging of the Kidney
Aging of Parenchymal and Stromal Compartments
The histological hallmarks of renal aging include glomerulosclerosis, tubular atrophy, interstitial fibrosis and arterial thickening . Thus, the aging process is not confined to the working parenchymal cells, but also involves the interstitial components of the kidney, including the vasculature ( Fig. 19.1 ). This is important because, although regenerating renal parenchyma may be the primary goal of regenerative therapy, its success will depend on the status of the extracellular matrix, stromal cells and the vasculature.
The capacity of the kidney parenchyma to regenerate is evident by the proliferative response of renal epithelial cells. Normally, the proportion of tubular cells proliferating at any given time is less than 1%. However, a burst of proliferation can occur in response to acute damage, a response that serves to regenerate the tubules . Importantly, this proliferative activity declines with age . Regenerative proliferation may also come from progenitor cell in niches within the renal papilla, Bowman’s capsule and proximal tubule , cells which will also be subjected to aging.
In association with this, several age-related changes in the extracellular matrix (ECM) may themselves impede the ability of parenchymal cells to replicate. These changes include increased collagen cross-linking, collagen glycation, and oxidation, nitration and racemization of various ECM or ECM-regulatory proteins . Studies on collagen glycation have been particularly informative in highlighting how the ECM can promote cellular aging. Endothelial cells cultured on glycated collagen display features of premature senescence, including senescence-associated β-galactosidase activity and upregulation of p53 and p14 ARF . Similar aging attributes are found in vivo in diabetic rats and a functional linkage with reduced nitric oxide (NO) availability has been found .
Aging of cellular elements of the interstitium can also be expected to impact the capacity of the kidney for regeneration. Mesenchymal stem cells (MSCs) can regulate regeneration in at least two ways. The classic stem cell paradigm may apply whereby MSCs divide occasionally to produce daughter cells that will produce committed lineages of mature differentiated cells. In this regard, kidney-derived mesenchymal cells have been found to differentiate towards endothelial and SMC lineages to support new blood vessel formation and juxtaglomerular cells , among other phenotypes. In addition, there is growing evidence that MSCs can operate outside this paradigm and contribute to remodeling as a nidus of paracrine signaling . Aging of MSCs will almost certainly compromise either paradigm. In general, there is a less permissive environment for mesenchymal cell remodeling with aging due to senescence of MSCs, telomere shortening and increased burden of reactive oxygen species (ROS) .
Aging of the Renal Vasculature
The aging vasculature can impact on renal function and its potential for regeneration in several ways. First, aged renal vasculature can lead to impaired autoregulation of the afferent and efferent arterioles. The resulting flow dysregulation can lead to hyperperfusion glomerular injury and expansion of the mesangial matrix. The capacity to regenerate glomeruli will thus depend on the extent to which this aspect of vascular function can be preserved.
Another form of age-related vascular dysfunction is arterial or arteriolar occlusive disease leading to ischemic injury. The resulting canonical damage cascade can drive both glomerular and tubulointerstitial pathology. Importantly, aging-related renal ischemia probably proceeds even in the absence of overt arterial occlusive disease. Studies in the rat kidney have identified aging-related increases in expression of key hypoxia-induced genes, including hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) .
A third relevant feature of the aging vasculature is increased stiffness . Arterial stiffness will increase pulse wave velocity and transmit uncompensated force to the distal microvasculature. This can have important consequences for renal function and it has been determined that albuminuria correlates with large artery stiffness .
Finally, emerging evidence suggests that vascular pericytes have putative roles as progenitor populations . Although this has yet to be demonstrated for pericytes of the renal vasculature, pericytes from diverse vascular beds have been found to have broad multipotency, suggesting another means by which the aging vasculature could compromise organ regeneration. Collectively, therefore, aging of the vasculature can have diverse adverse consequences for kidney function and restorative capacity.
Cellular Basis of Tissue Aging
The cellular basis of tissue aging is the subject of a number of excellent reviews and will not be reviewed in detail. However, one important paradigm of tissue aging is that of cellular senescence. Although best studied in culture, cell senescence is strongly implicated in age-related pathologies as well as the recognized decline in tissue regenerative potential with age .
Cellular senescence was first described by Hayflick and Moorfield, who observed that human fibroblasts replicating in culture eventually, and irreversibly, arrested their growth . Growth arrest during senescence appears to be a programmed event and normally occurs in the G1 phase of the cell cycle. Importantly, however, senescence is not a mode of death. Provided ambient conditions are satisfactory, senescent cells remain viable and can maintain metabolic activity indefinitely . Indeed, studies have revealed that senescent cells can be resistant to apoptosis and show reduced autophagy . However, senescent cells are not believed to be neutral with respect to tissue function. Their inability to replicate means that they cannot participate in reparative or homeostatic processes. Senescence of stem cells will similarly abrogate stem cell-based regeneration. In addition, senescent cells have been shown to be proinflammatory , further compromising tissue health.
Telomere-dependent and Telomere-independent Cell Senescence
One widely discussed hypothesis for explaining replicative senescence relates to telomeres. Telomeres are DNA–protein complexes at the ends of the chromosomes that protect the chromosomes from degradation, recombination and fusion . DNA replication at these chromosomal ends sites is semi-conservative, meaning that the duplication process is not complete. To overcome this, telomere length can be maintained by telomerase, a ribonucleoprotein that adds telomeric DNA to the chromosome, using the RNA of this complex as a template. In somatic cells, telomerase activity is too low for this telomere regeneration strategy to be effective. The result is that telomeres successively shorten with each cell division.
Critical telomere shortening produces telomere dysfunction, which is thought to trigger senescence. A primary pathway by which senescence is triggered is through the p53 tumor suppressor pathway. Telomere dysfunction can lead to ATM-dependent stabilization of p53 with resultant transactivation of p21. p21 expression, in turn, will inhibit cyclin-dependent kinase, one consequence of which will be hypophosphorylation of the retinoblastoma protein (Rb). Hypophosphorylated Rb remains bound to the E2F transcription factor and thereby imposes growth arrest. In the kidney, the role of telomerase has recently been elucidated using a mouse model. The regenerative capacity of acutely injured kidneys was found to be impaired in telomerase-deficient mice . These mice displayed both increased senescence and increased apoptosis. Although this differs from the classic notion that senescent cells are resistant to apoptosis, it also highlights the fact that there are multiple roles for the telomerase enzyme .
Senescence can also proceed in the absence of cell division and telomere shortening. In this case, irreversible growth arrest is triggered by one or more stresses and the term stress-induced premature senescence (SIPS) is applied. This form of senescence may be particularly relevant to the in vivo situation where cell proliferation rates may be low. Although cells subjected to SIPS do not display telomere attrition, they share many attributes with those subjected to replicative senescence and are thus deleterious to tissue function. One of the most physiologically relevant inducers of SIPS is oxidative stress. The accumulation of ROS is known to induce cumulative damage to mitochondrial DNA and membrane proteins during aging. Relevant signaling targets include promoters of genes encoding the tumor suppressors p16 INK4a and p 14ARF .
Molecular Mechanisms Underlying Cellular Aging
Lessons from Lower Organisms
Studies of lower organisms have proven to be a fruitful means of discovering pathways that may underlie human aging and senescence. One of the major advances was the discovery that the lifespan of yeast, worms, flies and rodents can be extended by caloric restriction . Although several pathways are likely to underlie this response, the process in general falls under the category of “what doesn’t kill you makes you stronger”. Thus, caloric restriction is believed to activate adaptive stress responses that evolved to help organisms to survive periods of starvation .
One of the molecular cascades that has been linked to this response involves a class of protein deacetylases known as sirtuins. Sirtuins remove the acetyl group from a number of target proteins through a unique two-step reaction . Although this may seem unnecessarily complex, this two-step deacetylation reaction requires the hydrolysis of NAD + , a requirement that ensures that the activity of sirtuins is regulated by NAD + availability. The importance of this level of regulation will be discussed below.
The best studied sirtuin in lower organisms is silent information regulator-2, or Sir2. In the absence of Sir2, yeast ( Saccharomyces cerevisiae ), worms ( Caenorhabditis elegans ) and flies ( Drosophila melanogaster ) no longer show extended longevity in response to nutrient restriction . Likewise, extra copies or increased expression of the Sir2 gene extends the lifespan of yeast, worms and flies . The relevant mechanisms by which Sir2 confers lifespan extension in these species include silencing of telomeres and inhibiting the activity of cell death proteins such as p53 and Ku70, through NAD + -dependent deacetylation . The extent to which these specific pathways translate to sirtuin function in humans is uncertain. However, as discussed below, mammalian sirtuins have diverse actions that can positively affect cellular health in several ways.
NAD + -consuming Reactions and Mammalian Cell Health
NAD + (the oxidized form of nicotinamide adenine dinucleotide) is classically known as a cofactor for the hydride transfer enzymes of intermediary metabolism . These well-known reactions entail the recycling of NAD + [or nicotinamide adenine dinucleotide phosphate (NADP)] between oxidized and reduced forms. However, in recent years, entirely unexpected roles for NAD + have been discovered. These new, but vital, roles are not based on redox events, but instead are characterized by NAD + consumption. This is because NAD + is not a cofactor for these reactions, but rather is a substrate that is enzymatically degraded.
Three classes of mammalian NAD + -consuming enzymes are currently recognized: (i) ADPribose transferases and poly(ADPribose) polymerases (PARPs), (ii) cADPribose synthases, and (iii) sirtuins ( Fig. 19.2 ). The wide range of reactions mediated by these enzymes includes DNA repair, chromatin silencing, transcriptional regulation, metabolic switching, calcium mobilization and lifespan regulation . Although all three classes perform indispensable functions within cells, only the sirtuins (particularly the founding member and mammalian homolog of Sir2, SIRT1) have been consistently implicated in mammalian cell survival and replicative longevity.
Sirtuins and Cellular Metabolism
There are seven known mammalian sirtuins, or SIRTs. SIRT1, 2, 3, 5 and 7 have deacetylase activity, consistent with Sir2 in lower organisms. SIRT4 and SIRT6 have ADP-ribosyltransferase activity which, like SIRT-mediated deacetylase activity, requires NAD + .
The best studied mammalian SIRT is SIRT1, an enzyme that impinges on an array of intracellular processes. One of the primary roles of SIRT1 is as a master regulator of metabolism, by virtue of its activity in fat, muscle, liver and pancreas. SIRT1 activity mobilizes fatty acids in white adipose tissue and prevents preadipocyte differentiation. These “fat-losing” processes proceed via SIRT1-mediated repression of peroxisome proliferator activated receptor-γ (PPAR-γ) interactions with its cofactors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptor (SMRT) . In skeletal muscle, SIRT1 deacetylates and activates PPAR-γ coactivator-1α (PGC-1α), which serves to increase fatty acid utilization and aerobic capacity . A similar activation of PGC-1α in the liver results in increased gluconeogenesis . Control of hepatic gluconeogenesis by SIRT1 is also conferred by deacetylation, and associated nuclear trapping, of the forkhead box transcription factor, FoxO1 .
Importantly, the SIRT1-mediated reduction in adipose tissue and enhanced skeletal muscle aerobic capacity together serve to increase insulin sensitivity . SIRT1 can also directly increase the action of insulin in these tissues by repressing protein tyrosine phosphatase 1B expression , a negative regulator of insulin signaling. In addition, SIRT1 enhances glucose-stimulated insulin secretion by the pancreas by repressing expression of uncoupling protein-2 and protects β-cells from glucose toxicity during hyperglycemia, through deacetylation of FoxO1 .
Although the salutatory effects of SIRT1 on the metabolic milieu have yet to be thoroughly evaluated in the context of renal disease, the importance of glucose control to renal performance, and of glucose and lipid metabolism to renovascular health, highlight the central role that SIRT1-mediated metabolic cascades may play in the kidney. Recent data have shown that the SIRT1 activator, resveratrol, ameliorates glucose-mediated dysfunction of glomerular epithelial cells . Thus, the data to date suggest that SIRT1 is critical to establishing a favorable metabolic milieu for tissue repair and regeneration.
Sirtuins and Endothelial Cells
Endothelial cell senescence is a feature of aging and vascular disease. In addition to growth arrest, senescent endothelial cells are characterized by decreased NO and prostacyclin production, increased plasminogen activator inhibitor-1 (PAI-1) expression and enhanced monocyte adhesion properties , phenomena that contribute substantially to the pathogenesis of vascular disease.
All seven SIRTs are expressed in human vascular endothelial cells . As with the metabolic cascades noted above, however, most studies of sirtuins and endothelial cells have focused on the role of SIRT1. There is an association between SIRT1 activity and NO metabolism. In vitro, SIRT1 has been found to deacetylate lysines 496 and 506 of endothelial nitric oxide synthase (eNOS), thereby stimulating its activity and the endothelial cell production of NO . Corresponding studies in mice subjected to caloric restriction confirmed enhanced deacetylation of eNOS in response to SIRT1 activation in vivo . Moreover, mice given low doses of red wine, the predominant source of the activator of SIRT1 resveratrol, show coordinate increases in SIRT1 and eNOS expression .
SIRT1 also plays a role in regulating endothelial cell longevity and resistance to senescence. Inhibition of SIRT1 expression and activity in human umbilical vein endothelial cells has been shown to induce premature senescence, characterized by increased PAI-1 expression and decreased expression and activity of eNOS . Consistent with this, overexpression of SIRT1 , activation of SIRT1 with resveratrol or induction of SIRT1 expression by the PDE3 inhibitor cilostazol prevented oxidative stress-induced premature senescence and the associated inflammatory phenotype in endothelial cells. Cilostazol appears to induce SIRT1 expression through an eNOS-dependent pathway , a mechanism previously identified in adipose tissue of mice subjected to caloric restriction .
SIRT1 also has angiogenic actions. Potente et al. demonstrated that NAD + -dependent deacetylation of FoxO1 by SIRT1 inhibits the antiangiogenic activity of FoxO1 in endothelial cells, and that neovascularization after acute hindlimb ischemic injury was blunted in endothelial cell-specific SIRT1-deficient mice . Moreover, studies of putative circulating human endothelial progenitor cells (EPCs) exposed to high glucose suggest that the resulting oxidative stress diminishes SIRT1 expression and activity, with concomitant increases in FoxO1 acetylation . Thus, SIRT1 may promote vascular regeneration by deacetylation pathways, in both mature and progenitor endothelial phenotypes, outcomes that would be expected to favor the restoration of optimal kidney function.
Sirtuins and Vascular Smooth Muscle Cells
Vascular smooth muscle cells (SMCs) are central to the regulation of blood flow as well as the repair of damaged vasculature. Deteriorated function of vascular SMCs with aging can have profound effects on tissue homeostasis and regenerative potential. As one important example, the efficiency with which SMCs stabilize atherosclerotic lesions can determine whether the lesion will rupture, a potentially fatal event. Vascular SMC senescence has recently been identified as a feature of atherosclerotic lesions . Senescent SMCs may be particularly dangerous because the resulting proinflammatory and non-reparative state could incite lesion disruption and acute vascular occlusion. Strategies to prevent the premature senescence of SMCs could thus be a promising approach for reducing vascular insufficiency of regenerating organs, including the kidney, if molecular targets can be identified.
A role for SIRT1 in regulating SMC senescence is emerging, although it appears to be context dependent. As noted, SIRT1 has been reported to inhibit senescence in endothelial cells and a similar response has been observed for human cancer cells and fibroblasts . However, in other cells SIRT1 has also been reported to promote senescence . In still other studies SIRT1 was found to have no effect on senescence at all . One study found that overexpression of SIRT1 in SMCs led to a modest reduction in the accumulation of senescent cells and a modest extension in SMC replicative longevity . However, SMC lifespan was markedly extended when SIRT1 was overexpressed together with nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme for the NAD + salvage pathway ( Fig. 19.3 , and discussed further below) . Furthermore, it was found that SIRT1 activity declined, concomitant with the capacity to regenerate NAD + , as SMCs underwent replicative aging. Thus, understanding the role of SIRT1 in human cell aging, and the potential benefits of activating SIRT1 for tissue regeneration, must take into account the prevailing metabolic conditions in which the enzyme operates.