Senescence-associated phenotypes in Akita diabetic mice are enhanced by absence of bradykinin B2 receptors

Abstract

We have previously reported that genetically increased angiotensin-converting enzyme levels, or absence of the bradykinin B2 receptor, increase kidney damage in diabetic mice. We demonstrate here that this is part of a more general phenomenon - diabetes and, to a lesser degree, absence of the B2 receptor, independently but also largely additively when combined, enhance senescence-associated phenotypes in multiple tissues. Thus, at 12 months of age, indicators of senescence (alopecia, skin atrophy, kyphosis, osteoporosis, testicular atrophy, lipofuscin accumulation in renal proximal tubule and testicular Leydig cells, and apoptosis in the testis and intestine) are virtually absent in WT mice, detectable in B2 receptor-null mice, clearly apparent in mice diabetic because of a dominant mutation (Akita) in the Ins2 gene, and most obvious in Akita diabetic plus B2 receptor-null mice. Renal expression of several genes that encode proteins associated with senescence and/or apoptosis (TGF-beta1, connective tissue growth factor, p53, alpha-synuclein, and forkhead box O1) increases in the same progression. Concomitant increases occur in 8-hydroxy-2'-deoxyguanosine, point mutations and deletions in kidney mitochondrial DNA, and thiobarbituric acid-reactive substances in plasma, together with decreases in the reduced form of glutathione in erythrocytes. Thus, absence of the bradykinin B2 receptor increases the oxidative stress, mitochondrial DNA damage, and many senescence-associated phenotypes already present in untreated Akita diabetic mice.

Figure 1. Appearance and associated indicators of senescence in mice at age 12 months.

(A) General appearance of male mice with the following genotypes: WT at both the insulin 2 and the bradykinin B2 receptor loci, homozygous null for the B2 receptor (Bdkrb2^–/–), heterozygous Akita for insulin 2 (Ins2^Akita/+), and doubly mutant (Bdkrb2^–/–Ins2^Akita/+). Kyphosis was moderate in the Akita diabetic mouse; it was severe in the doubly mutant animal, which also exhibited marked alopecia. The animals imaged were among the most severely affected of each genotype. (B) H&E-stained abdominal skin of mice with the same 4 genotypes. Scale bars: 1 mm. Subcutaneous fat (asterisks) was present in both nondiabetic animals but was absent in the diabetic animals. The skin of the double mutant was thin and had few hair follicles. The areas selected for imaging are representative of each genotype. (C) Bone mineral density of femurs of mice of the 4 genotypes, assessed with dual-emission x-ray absorptiometry. Data are presented as means ± SEM with the numbers of animals shown in white digits (see Supplemental Table 4 for 2-factor ANOVA analysis).

Figure 2. Testes of 12-month-old mice of the 4 genotypes.

(A) Low-magnification H&E-stained testes. Loss of spermatogonia was severe in the double mutant. Some spermatic cords in the Akita diabetic mouse were aspermic (outside the dotted red line). The testes of the B2 receptor–null and WT mice had normal spermatic cords. (B) High-magnification H&E-stained testes of 12-month-old mice. There were numerous intracellular pigmented vacuoles in the Leydig cells of the doubly mutant mouse. Pigmented vacuoles in Leydig cells were also present to a somewhat lesser degree in the Akita diabetic mouse. They were sparse in the B2-null mouse, and absent in the WT mouse. The areas selected for imaging in A and B are representative of each genotype. Scale bars: 100 μm (A), 10 μm (B).

Figure 3. Kidneys of 12-month-old mice of the 4 genotypes.

(A) PAS-stained kidneys. There were numerous intracellular pigmented vacuoles in the cytoplasm of the proximal tubular epithelial cells of the doubly mutant mouse. Pigmented vacuoles were present to a lesser degree in the proximal tubules of the Akita diabetic mouse with intact B2 receptors but were absent in the B2 receptor–null and WT nondiabetic mice. The areas selected for imaging are representative of each genotype. (B) Transmission electron micrograph of a renal proximal tubule cell from a doubly mutant mouse. Note the numerous lipid debris–containing phagolysosomes with focal laminations (arrows). Scale bars: 10 μm (A), 1 μm (B).

Figure 4. Indices of oxidative stress in 12-month-old mice of the 4 genotypes.

(A) Plasma levels of TBARSs. (B) Concentration of reduced glutathione (GSH) in erythrocytes. Data are presented as means ± SEM with the numbers of animals shown in white digits (see Supplemental Table 4 for 2-factor ANOVA analysis).

Figure 5. Alterations in mtDNA in 12-month-old mice of the 4 genotypes.

(A) 8-OHdG content in renal cortical mtDNA. (B) Frequencies of point mutations in the cytochrome b gene in the mtDNA used in A. (C) Relative proportion of D-17 deletions (as percentage of WT) in the same DNA, determined by quantitative PCR. Data are presented as means ± SEM with the numbers of animals shown in white digits (see Supplemental Table 4 for 2-factor ANOVA analysis).

Figure 6. Oxidative stress in vitro.

(A) Mitochondrial oxidation (assessed by MitoTracker Red CM-H₂XRos fluorescence) in EA.hy926 vascular endothelial cells cultured in the presence of normal glucose (NG), high glucose (HG), HG plus 1 μM bradykinin (BK), or HG plus BK plus 1 mM l-NAME. BK reduced the oxidative signal seen in HG; this reduction was largely abolished by the NOS inhibitor l-NAME. Scale bars: 100 μm. (B) Quantitative analysis of MitoTracker Red fluorescence from single cells under the 4 culture conditions used in A. The data are means ± SEM with the numbers of cells shown in white digits.