Angiotensin II Type 1 Receptor-Associated Protein Regulates Kidney Aging and Lifespan Independent of Angiotensin

Abstract

Background: The kidney is easily affected by aging-associated changes, including glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Particularly, renal tubulointerstitial fibrosis is a final common pathway in most forms of progressive renal disease. Angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP), which was originally identified as a molecule that binds to AT1R, is highly expressed in the kidney. Previously, we have shown that ATRAP suppresses hyperactivation of AT1R signaling, but does not affect physiological AT1R signaling.

Methods and results: We hypothesized that ATRAP has a novel functional role in the physiological age-degenerative process, independent of modulation of AT1R signaling. ATRAP-knockout mice were used to study the functional involvement of ATRAP in the aging. ATRAP-knockout mice exhibit a normal age-associated appearance without any evident alterations in physiological parameters, including blood pressure and cardiovascular and metabolic phenotypes. However, in ATRAP-knockout mice compared with wild-type mice, the following takes place: (1) age-associated renal function decline and tubulointerstitial fibrosis are more enhanced; (2) renal tubular mitochondrial abnormalities and subsequent increases in the production of reactive oxygen species are more advanced; and (3) life span is 18.4% shorter (median life span, 100.4 versus 123.1 weeks). As a key mechanism, age-related pathological changes in the kidney of ATRAP-knockout mice correlated with decreased expression of the prosurvival gene, Sirtuin1. On the other hand, chronic angiotensin II infusion did not affect renal sirtuin1 expression in wild-type mice.

Conclusions: These results indicate that ATRAP plays an important role in inhibiting kidney aging, possibly through sirtuin1-mediated mechanism independent of blocking AT1R signaling, and further protecting normal life span.

Keywords: aging; chronic kidney disease; fibrosis; renin angiotensin system.

Figure 1

ATRAP deficiency does not affect external signs of aging. A, Senescence‐related changes in body weight were similar in ATRAP‐KO and WT mice. Values are expressed as mean±SE (n=6–8). ^# P<0.01, 3 to 4 months old vs 11 to 12 months old, **P<0.01, 3 to 4 months old vs 22 to 25 months old. B, There was no difference in gross appearance between aged ATRAP‐KO and WT mice. Mice exhibited similar signs of senescence, such as gray and white hair as well as some hair loss. ATRAP indicates angiotensin II type 1 receptor‐associated protein; KO mice, angiotensin II type 1 receptor‐associated protein‐knockout mice; WT mice, wild‐type mice.

Figure 2

ATRAP deficiency does not affect cardiovascular aging phenotype. A, Representative images of hematoxylin and eosin–stained sections of hearts of aged ATRAP‐KO and WT mice (top: original magnification, ×40; bar, 1 mm), and representative images of Masson's trichrome–stained sections of hearts of aged ATRAP‐KO and WT mice (bottom: original magnification, ×200; bar, 100 μm). B, Ratio of heart weight/body weight was similar in aged ATRAP‐KO and WT mice. Values are expressed as mean±SE (n=6–8). C, Cardiac fibrosis was comparable between aged ATRAP‐KO and WT mice. Values are expressed as mean±SE (n=6–8). D, Cardiac BNP mRNA expression was similar in aged ATRAP‐KO and WT mice. Values are expressed as mean±SE (n=6–8). E, Representative images of hematoxylin and eosin–stained sections of aortas of aged ATRAP‐KO and WT mice (top: original magnification, ×100; bar, 100 μm), representative images of Masson's trichrome–stained sections of aortas of aged ATRAP‐KO and WT mice (middle: original magnification, ×200; bar, 100 μm), and representative images of Elastica van Gieson‐stained sections of aortas of aged ATRAP‐KO and WT mice (bottom: original magnification, ×200; bar, 100 μm). F, Aortic wall thickness was comparable between ATRAP‐KO and WT mice. Values are expressed as mean±SE (n=6–8). ATRAP indicates angiotensin II type 1 receptor‐associated protein; BNP, brain natriuretic peptide; KO mice, angiotensin II type 1 receptor‐associated protein‐knockout mice; WT mice, wild‐type mice.

Figure 3

ATRAP deficiency exacerbates renal fibrosis by senescence. A, Representative images of hematoxylin and eosin–stained sections of kidneys of young and aged ATRAP‐KO and WT mice (original magnification, ×40; bar, 1 mm). B, Ratio of kidney weight/body weight was comparable between age‐matched ATRAP‐KO and WT mice. Values are expressed as mean±SE (n=6–8). **P<0.01, young vs aged mice. C, Representative images of hematoxylin and eosin–, PAS‐, and Masson's trichrome–stained sections of kidneys of young and aged ATRAP‐KO and WT mice (original magnification, ×200; bar, 100 μm). D, Glomerular sclerosis index was significantly and similarly increased in both aged ATRAP‐KO and WT mice compared with young groups. Values are expressed as mean±SE (n=6–8). **P<0.01 vs young. E, Renal fibrotic area was significantly increased in aged ATRAP‐KO mice compared with aged WT mice. Values are expressed as mean±SE (n=6–8). **P<0.01 vs young; ^‡ P<0.01 vs WT mice. ATRAP indicates angiotensin II type 1 receptor‐associated protein; HE, hematoxylin and eosin; KO mice, angiotensin II type 1 receptor‐associated protein‐knockout mice; PAS, periodic acid–Schiff; WT mice, wild‐type mice.

Figure 4

ATRAP deficiency enhances upregulation of renal collagen‐3α and transforming growth factor‐β (TGF‐β) mRNA expression by senescence. Renal mRNA expression of fibrosis‐related factors (A, collagen‐1α; B, collagen‐3α; C, TGF‐β; D, TNF‐α) in young and aged ATRAP‐KO and WT mice. Values are expressed as mean±SE (n=6–11). *P<0.05 vs young; **P<0.01 vs young; ^‡ P<0.01 vs WT mice. ATRAP indicates angiotensin II type 1 receptor‐associated protein; KO mice, angiotensin II type 1 receptor‐associated protein‐knockout mice; TGF‐β, transforming growth factor‐β; TNF‐α, tumor necrosis factor‐α; WT mice, wild‐type mice.

Figure 5

ATRAP deficiency provokes renal mitochondrial abnormalities and exacerbates oxidative stress. A, Representative electron microscopy images of renal proximal tubules in aged ATRAP‐KO and WT mice (original magnification, ×5000; bar, 500 nm). Proximal tubular cells of aged ATRAP‐KO mice exhibited an evident decrease in normal mitochondria morphology as well as an increase in abnormal mitochondria, with swelling and disintegration of the cristae. B, Renal mRNA expression of mitochondrial functions (UCP2, PGC‐1α, and Bnip3) in aged ATRAP‐KO and WT mice. Values are expressed as mean±SE (n=6–8). ^‡ P<0.01 vs WT mice. C, Renal protein expression of 4‐HNE in aged ATRAP‐KO and WT mice. Values are expressed as mean±SE (n=6–8). ^† P<0.05 vs WT mice. D, Representative immunohistochemistry for 4‐HNE (top) and quantitative analysis (bottom) in kidney sections of aged ATRAP‐KO and WT mice. Areas positive for 4‐HNE are evident as brown dots in sections (original magnification, ×100; bar, 100 μm). Values are expressed as mean±SE (n=6–8). ^† P<0.05 vs WT mice. 4‐HNE indicates 4‐hydroxy‐2‐nonenal; ATRAP, angiotensin II type 1 receptor‐associated protein; Bnip3, B‐cell lymphoma 2/adenovirus E1B 19‐kDa interacting protein 3; KO mice, angiotensin II type 1 receptor‐associated protein‐knockout mice; PGC‐1α, peroxisome proliferator‐activated receptor γ coactivator‐1α; UCP2, uncoupling protein 2; WT mice, wild‐type mice.

Figure 6

ATRAP deficiency provokes downregulation of renal SIRT1 protein expression. A, Renal protein expression of prosurvival factors (SIRT1, SIRT3, and Nampt) in young and aged ATRAP‐KO and WT mice. Values are expressed as mean±SE (n=6–8). *P<0.05 vs young; ^† P<0.05 vs WT mice. B, Immunohistochemistry for SIRT1 protein in kidney sections of ATRAP‐KO and WT mice. Areas positive for SIRT1 are evident as brown dots in sections (original magnification, ×200; bar, 100 μm). ATRAP indicates angiotensin II type 1 receptor‐associated protein; KO mice, angiotensin II type 1 receptor‐associated protein‐knockout mice; Nampt, nicotinamide phosphoribosyltransferase; NS, nonspecific band; SIRT1, sirtuin1; SIRT3, sirtuin3; WT mice, wild‐type mice.

Figure 7

Activation of the Ang II–AT1R axis by chronic Ang II infusion does not reduce renal SIRT1 in young WT mice. Renal protein expression of prosurvival factors (SIRT1, SIRT3, and Nampt) in Ang II– or vehicle‐infused young WT mice. Values are expressed as mean±SE (n=6–8). ^§ P<0.05 vs vehicle; ^¶ P<0.01 vs vehicle. Ang II indicates angiotensin II; ATRAP, angiotensin II type 1 receptor‐associated protein; Nampt, nicotinamide phosphoribosyltransferase; SIRT1, sirtuin1; SIRT3, sirtuin3; WT mice, wild‐type mice.

Figure 8

ATRAP deficiency does not enhance renal CAML and NFAT signaling pathway by senescence. Renal mRNA expression of factors in the CAML‐NFAT signaling pathway (A, CAML; B, NFAT1; C, NFAT3; D, NFAT4; E, IL‐2) in young and aged ATRAP‐KO and WT mice. Values are expressed as mean±SE (n=6–8). *P<0.05 vs young; **P<0.01 vs young; ^† P<0.05 vs WT mice; ^‡ P<0.01 vs WT mice. ATRAP indicates angiotensin II type 1 receptor‐associated protein; CAML, calcium‐modulating cyclophilin ligand; IL‐2, interleukin‐2; KO mice, angiotensin II type 1 receptor‐associated protein‐knockout mice; NFAT1, cytoplasmic, calcineurin‐dependent 2 (NFATc2); NFAT3, nuclear factor of activated T cells, cytoplasmic, calcineurin‐dependent 4 (NFATc4); NFAT4, nuclear factor of activated T cells, cytoplasmic, calcineurin‐dependent 3 (NFATc3); WT mice, wild‐type mice.

Figure 9

ATRAP deficiency shortens life span. Cumulative survival of ATRAP‐KO mice (n=21) and WT mice (n=23) was analyzed using the Kaplan–Meier method. P=0.0002 vs WT mice by the log‐rank test. Median life spans of ATRAP‐KO mice and WT mice were 100.4 and 123.1 weeks, respectively. ATRAP indicates angiotensin II type 1 receptor‐associated protein; KO mice, angiotensin II type 1 receptor‐associated protein‐knockout mice; WT mice, wild‐type mice.