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Dogs exhibit striking within-species variability in lifespan, with smaller breeds often living more than twice as long as larger breeds. This longevity discrepancy also extends to health and aging-larger dogs show higher rates of age-related diseases. Despite this well-established phenomenon, we still know little about the biomarkers and molecular mechanisms that might underlie breed differences in aging and survival. To address this gap, we generated an epigenetic clock using DNA methylation from over 3 million CpG sites in a deeply phenotyped cohort of 864 companion dogs from the Dog Aging Project, including some dogs sampled annually for 2-3 years. We found that the largest breed size tends to have epigenomes that are, on average, 0.37 years older per chronological year compared to the smallest breed size. We also found that higher residual epigenetic age was significantly associated with increased mortality risk, with dogs experiencing a 34% higher risk of death for each year increase in residual epigenetic age. These findings not only broaden our understanding of how aging manifests within a diverse species but also highlight the significant role that demographic factors play in modulating the biological mechanisms underlying aging. Additionally, they highlight the utility of DNA methylation as both a biomarker for healthspan-extending interventions, a mortality predictor, and a mechanism for understanding inter-individual variation in aging in dogs.

Our understanding of age-related physiology and metabolism has grown through the study of systems biology, including transcriptomics, single-cell analysis, proteomics and metabolomics. Studies in lab organisms in controlled environments, while powerful and complex, fall short of capturing the breadth of genetic and environmental variation in nature. Thus, there is now a major effort in geroscience to identify aging biomarkers and to develop aging interventions that might be applied across the diversity of humans and other free-living species. To meet this challenge, the Dog Aging Project (DAP) is designed to identify cross-sectional and longitudinal patterns of aging in complex systems, and how these are shaped by the diversity of genetic and environmental variation among companion dogs. Here we surveyed the plasma metabolome from the first year of sampling of the Precision Cohort of the DAP. By incorporating extensive metadata and whole genome sequencing information, we were able to overcome the limitations inherent in breed-based estimates of genetic and physiological effects, and to probe the physiological and dietary basis of the age-related metabolome. We identified a significant effect of age on approximately 40% of measured metabolites. Among other insights, we discovered a potentially novel biomarker of age in the post-translationally modified amino acids (ptmAAs). The ptmAAs, which can only be generated by protein hydrolysis, covaried both with age and with other biomarkers of amino acid metabolism, and in a way that was robust to diet. Clinical measures of kidney function mediated about half of the higher ptmAA levels in older dogs. This work identifies ptmAAs as robust indicators of age in dogs, and points to kidney function as a physiological mediator of age-associated variation in the plasma metabolome.