Aging is a degenerative biologic process that affects multiple organs or tissues including the kidneys. The aging-related decline in renal function is attributed, in part, to a progressive loss of functioning nephrons which eventually results in a decrease in glomerular filtration rate (GFR). The present study further demonstrated that the aged animals were associated with glomerulus collapse and glomerular and tubulointerstitial fibrosis. These changes would impair glomerular and tubular function, systemic hemodynamics, and, as a whole, body homeostasis, contributing to the aging process. The renal function may be damaged in the Old Impaired animals as evidenced by a significant increase in serum creatinine. Glomerulosclerosis is characterized by excessive accumulation of extracellular matrix in the kidney glomerulus. The glomerulosclerotic process is usually progressive in nature, eventually impairing the function of neighboring structures due to the gradual expansion of scar tissue and the collapse of glomeruli. The degree of glomerulosclerosis correlates with a progressive loss of renal function, and eventually end-stage renal disease (Taguchi et al. 2005
The cognitive performance declines with increasing age. Interestingly, the aged rats with impaired cognition showed exaggerated glomerular collapse (atrophy). These findings suggest, for the first time, that the aging-related kidney damage (e.g., glomerusclerosis) parallels with the cognitive function impairment. One possibility is that the same aging mechanism may affect both brain and kidneys in some aged animals. For instance, overactivation of the endothelin system may damage both brain and kidneys by impairing microvascular function in these organs (Zhang et al. 1994
; Kuiper et al. 2008
; Seccia et al. 2008
; Soleman et al. 2010
). On the other hand, the impaired kidney function results in accumulation of metabolic toxic products which could impair brain function (e.g., cognition). The mechanistic link of the aging-related kidney damage and cognitive impairment is an interesting topic to pursue.
Klotho is a recently-discovered anti-aging gene (Kuro-o et al. 1997
; Wang and Sun 2009a
). Klotho protein is predominantly expressed in renal tubule epithelial cells (Wang and Sun 2009a
). A decrease in renal klotho expression has been found in kidneys of hypertensive animals (Aizawa et al. 1998
; Wang and Sun 2009b
). The klotho production was severely reduced in human chronic failure kidneys (Koh et al. 2001
). Klotho deficiency causes kidney damage (Aizawa et al. 1998
; Koh et al. 2001
; Wang and Sun 2009b
), suggesting that klotho is essential in maintaining normal kidney structure and function. Interestingly, in vivo expression of klotho in kidneys prevents renal damage in spontaneous hypertensive rats (Wang and Sun 2009b
) and ameliorates angiotensin II-induced renal injury (Mitani et al. 2002
). Klotho reduces apoptosis in acute experimental ischemic renal failure (Sugiura et al. 2005
). Genetic manipulation of klotho ameliorates progressive renal injury (Haruna et al. 2007
). Klotho increases nitric oxide availability and SOD activity (Wang and Sun 2009a
). Klotho gene delivery suppresses superoxide production in kidneys (Wang and Sun 2009b
). There are two forms of klotho, the membrane-form (renal) klotho and the secreted form (circulating) klotho due to alternative RNA splicing (Wang and Sun 2009a
). The present study demonstrated that klotho protein expression was downregulated in kidneys while the circulating klotho level remained unchanged in aged rats. This finding is new because it may open a new avenue for studying the aging-related kidney damage. An impairment in the RNA splicing for generating the membrane-form klotho could lead to a decrease in the renal klotho, but the exact mechanism remains to be found. The tissue level of klotho may be more important than the circulating klotho in determining its function in the kidney. The present study, however, cannot determine if klotho is involved in kidney damage. Therefore, a further study is required to assess if the downregulation of renal klotho expression contributes to the aging-related kidney damage by in vivo kidney-specific expression of klotho in aged animals.
The action of ET-1 is mediated by ETa and ETb receptors. Activation of ETa receptors increases NADPH oxidase activity and superoxide production (Dammanahalli and Sun 2008b
) which may contribute to aging-related kidney damage. In contrast, activation of ETb receptors increases the NO release in kidney medulla (Abassi et al. 2001
) which may protect the kidneys. Therefore, the effects of ET-1 in kidney depend on the location and regulation of ETa and ETb receptors. Although aging-related changes in ET-1 and ET receptors were reported in hearts and bladders (Afiatpour et al. 2003
; Yono et al. 2004
; Del Ry et al. 2008
), the regulation of ET receptors in the aged kidneys was poorly studied. Lattmann et al. reported that both ETa and ETb mRNA levels were decreased in aged kidneys (Lattmann et al. 2005
). The present finding revealed that ETa receptor protein expression was upregulated in renal cortex and medulla while ETb receptor protein expression was downregulated in medulla. Interestingly, the ratio of ETa:ETb was increased in renal medulla in aged rats although the level of the circulating ET-1 was not altered. These changes may contribute to the aging-related kidney damage. The suppression of medullary ETb expression was more pronounced in the Old Impaired animals, which may contribute to the aggravation of the kidney damage in this group.
Another important finding is that kidney aging was associated with increased inflammation as evidenced by increased IL-6 expression in both glomeruli and tubules in aged rats. The exacerbated kidney damage in the Old Impaired rats may be attributed, in part, to aggravated inflammation because the aging-related increase in IL-6 expression was more pronounced in the Old Impaired rats. Inflammation could activate TGF-β leading to tissue remodeling (Izad et al. 2010
; Wang et al. 2010
). The mechanism of aging-related increase in IL-6 is not fully understood. IL-6 is secreted from macrophages (Suzuki et al. 2004
) and is the main cytokine involved in the induction of inflammation (Czarkowska-Paczek et al. 2005
). It was reported that IL-6 expression was increased with age in rats (Franchini and Ottaviani 2007
) and humans (Friedman et al. 2007
). Its production is enhanced by renal mesangial cells, which may be associated with mesangial cell proliferation or interstitial tissue injury as proposed by Suzuki et al. (Suzuki et al. 1995
). These cells adhere to the glomerular capillary wall, between endothelial cells and the basal lamina. The present data showed significant mesangial expansion based on the PAS staining. IL-6 protein expression was increased in the peritubular spaces in renal medulla of aged rats, suggesting that inflammation may be involved in interstitial fibrosis.
Cumulative oxidative injury is believed to play a major role in the process of cell aging. Oxidative stress and generation of free radicals increase with aging (Mendoza-Nunez et al. 2007
). Persistent oxidative damage to cytosolic structures leads to cross-linking of oxidized proteins and deposition of lipofuscin with functionally and structurally impaired mitochondria (Jung et al. 2007
). Recent data suggest that chronic oxidant stress plays a contributory role in telomere shortening and thus in senescent changes (Houben et al. 2008
). The mechanism of aging-related kidney damage is not fully understood. In a rat model, kidney aging was associated with a 60% decline in GFR, a threefold increase in renal F2 isoprostanes, and an increase in oxidant-sensitive heme oxygenase (Thomas et al. 1998
). The NADPH oxidases may be involved in the pathogenesis of age-associated diseases (Krause 2007
). The level of oxidative stress is determined by superoxide production and dismutation. The present study demonstrated that NADPH oxidase Nox2 expression and superoxide production were increased in kidneys of aged animals, which could contribute to aging-related oxidative stress.
On the other hand, MnSOD expression was suppressed in kidneys of the Old Impaired rats which may contribute to the aging-related kidney damage. The mitochondria-derived superoxide is the major source of reactive oxygen species. MnSOD is the primary anti-oxidant enzyme that scavenges superoxide formed within the mitochondria and protects against oxidative stress. Overexpression of MnSOD protects against hyperglycemia-induced renal injury using normal rat renal proximal tubular cells (NRK) (Munusamy and MacMillan-Crow 2009
). The present study demonstrated that NOX-2 protein expression was upregulated but the MnSOD protein expression was downregulated in renal cortex and medulla of aged rats. These changes increase the superoxide level contributing to oxidative injury in kidney aging.
The aging-related kidney damage may involve multiple molecular pathways. The present study revealed aging-related changes in klotho, ET receptors, Nox2, and MnSOD, providing a basis for assessing the roles and the relationship of these factors in kidney aging. ET-1 stimulates the production of superoxide via activation of NADPH oxidases (Dammanahalli and Sun 2008b
). On the other hand, NADPH oxidase-derived ROS appear to stimulate ET-1 production (Dammanahalli and Sun 2008b
). A decrease in klotho expression was associated with an increase in Nox2 expression and superoxide production in kidneys in spontaneous hypertensive rats (SHRs) (Wang and Sun 2009b
). Oxidative stress may decrease expression of klotho in a mouse inner medullary collecting duct (mIMCD3) cell line (Mitobe et al. 2005
). Klotho gene delivery suppressed Nox2 expression and superoxide production and attenuated kidney damage in SHRs (Wang and Sun 2009b
). There is growing evidence that the klotho protein induces the expression of MnSOD (Kuro-o 2008
) and suppression of NADPH oxidases (Wang and Sun 2009b
; Wang and Sun 2009a
) to protect against oxidative stress. Genetic mutation of klotho decreases SOD expression while overexpression of klotho increases SOD expression (Wang and Sun 2009a
), indicating that klotho may regulate SOD expression. Klotho gene delivery enhanced IL-10 level in the blood in SHRs (Wang and Sun 2009b
), suggesting that klotho may suppress inflammation. It is expected that a decrease in klotho may upregulate IL-6 in kidneys. This hypothesis, however, needs to be tested.
In summary, the aging-related kidney damage was associated with downregulation of klotho, ETb and MnSOD expression and upregulation of ETa, IL-6, and Nox2 expression and superoxide production. These changes were more pronounced in the Old Impaired animals. The findings raise the necessity to evaluate the roles of these factors and their relationship in aging-related kidney damage. The novel aspect of this study is that it reveals that the aging-related cognitive impairment paralleled with the kidney damage and that kidney aging was associated with a reduction of klotho.