Investigating the Mechanisms of Slowed Kidney Fibrosis via Calorie Restriction

The practice of calorie restriction produces sweeping changes in the operation of cellular metabolism and acts to modestly slow the progression of aging, extending life in most species and lineages tested to date. The focus in this open access paper is on the ability of calorie restriction to slow the fibrosis that accompanies aging, here in the kidney, though it is significant in other organs as well. Fibrosis is the inappropriate formation of scar-like tissue due to age-related failure in mechanisms of regeneration, a process that degrades organ function and is a major component of conditions such as kidney failure.

Chronic kidney disease (CKD), which is defined by reduced glomerular filtration rate, proteinuria, or structural kidney disease, is a growing problem among the aging population, to the extent that the elderly have an average prevalence of CKD that is three to five times higher than that observed in young and middle-aged populations. Accordingly, CKD predisposes the elderly to a high risk of cardiovascular events and premature death. Morphological and functional changes that accompany kidney aging are thought to contribute the development of CKD in the elderly. For example, processes that characterize kidney aging include glomerulosclerosis, interstitial fibrosis, tubular atrophy, vascular sclerosis, and loss of renal function. The mechanistic basis of kidney aging is cellular senescence, which is characterized by the inability of cells to proliferate despite the presence of ample space, nutrients and growth factors in the medium. Although renal fibrosis has been observed in elderly individuals in the absence of overt CKD, the relationship between cellular senescence and fibrosis during kidney aging is yet to be determined.

Epithelial-mesenchymal transition (EMT) is the process whereby differentiated epithelial cells undergo a phenotypic conversion that gives rise to matrix-producing fibroblasts and myofibroblasts. EMT is increasingly recognized as a key process that contributes to kidney fibrosis and the decline of renal function. Age-related changes in the levels of transforming growth factor-β (TGF-β), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) result in a complex shift of the microenvironmental milieu that is thought to affect tissue homeostasis under both normal and abnormal conditions, triggering EMT and progressive fibrosis. Given that senescence and EMT play well-documented roles in the etiology and progression of age-associated CKD, the development of therapeutic interventions to retard or reverse these processes is warranted.

Here, we first examined compared age-related metabolic parameters and renal function between the groups of rats. Compared to the young rats, increases in age-related proteinuria, hypercholesterolemia and hypertriglyceridemia were observed in the older rats. We proceeded to demonstrate increased cellular senescence, as indicated by overexpression of P16, P21 and SA-β-gal in the kidneys of the aging rats. Cellular senescence is not only a marker of renal aging but also actively participates in the process. Furthermore, senescent cells secrete inflammatory factors and growth factors, resulting in a complex shift within the cellular microenvironment, which induces EMT. Increased levels of EMT as a function of age were demonstrated in this study. We found that EMT was increased in the older rats, compared with the younger rats, indicating that EMT increases as a function of age. Given that cellular senescence and EMT contribute to the decline in renal function with age, we investigated the effect of interventions on both senescence and age-related EMT in our rat models.

While previous studies have shown that caloric restriction (CR) decreased the abundance of senescent cells, improved telomere maintenance and reduced the levels of oxidative damage markers in the small intestine and liver, and alleviated age-related increase in EMT in the thymus, similar studies in the aging kidney have been lacking. Here we report that short-term CR alleviates cellular senescence and EMT in the aging kidney. However, it should be pointed out that even were our study to substantiate short-term CR as an effective intervention for cell senescence and EMT, the degree of restriction required would limit the utility of this intervention. As an alternative strategy, new research has focused on the development of caloric restriction mimetics (CRMs). The objective of CRM research is to identify compounds that mimic the effects of CR by targeting metabolic and stress response pathways affected by CR without actually restricting caloric intake.

With respect to how short-term CR and CRM treatment might directly impact cellular senescence and EMT, one interesting candidate is the AMPK-mTOR signaling pathway. In the in vivo experiments, we demonstrated that AMPK/mTOR signaling in kidney was downregulated with age, and that this was reversed by short-term CR and CRM treatment. In order to further verify this pathway, we induced EMT and cellular senescence of proximal tubular cells (PTCs) in vitro with high glucose. We found that exposure of PTCs to high glucose for 48 hours resulted in the high glucose-induced EMT and cellular senescence, decreased expression of activated AMPK and decreased AMPK/mTOR signaling. Costimulation of PTCs with high glucose and a CRM, both of which activate AMPK, alleviated high glucose-induced EMT and cellular senescence, and increased AMPK/mTOR signaling. Moreover, mTOR was upregulated, and EMT and senescence were increased in AMPK-silenced cells, but were not alleviated in AMPK-silenced cells that had been treated with a CRM. These results indicated that the CRM inhibited EMT and senescence of PTC via AMPK/mTOR signaling. It is possible that the data presented here could be extrapolated to explain the mechanisms of fibrosis seen in other organs during aging, and to provide strategies to overcome this process.



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