Age-dependent renal cortical microvascular loss in female mice

Abstract

Renal function and blood flow decline during aging in association with a decrease in the number of intrarenal vessels, but if loss of estrogen contributes to this microvascular, rarefaction remains unclear. We tested the hypothesis that the decreased renal microvascular density with age is aggravated by loss of estrogen. Six-month-old female C57/BL6 mice underwent ovariectomy (Ovx) or sham operation and then were allowed to age to 18-22 mo. Another comparable group was replenished with estrogen after Ovx (Ovx+E), while a 6-mo-old group served as young controls. Kidneys were then dissected for evaluation of microvascular density (by micro-computed tomography) and angiogenic and fibrogenic factors. Cortical density of small microvessels (20-200 μm) was decreased in all aged groups compared with young controls (30.3 ± 5.8 vessels/mm², P < 0.05), but tended to be lower in sham compared with Ovx and Ovx+E (9.9 ± 1.7 vs. 17.2 ± 4.2 and 18 ± 3.0 vessels/mm², P = 0.08 and P = 0.02, respectively). Cortical density of larger microvessels (200-500 μm) decreased only in aged sham (P = 0.04 vs. young control), and proangiogenic signaling was attenuated. On the other hand, renal fibrogenic mechanisms were aggravated in aged Ovx compared with aged sham, but blunted in Ovx+E, in association with downregulated transforming growth factor-β signaling and decreased oxidative stress in the kidney. Therefore, aging induced in female mice renal cortical microvascular loss, which was likely not mediated by loss of endogenous estrogen. However, estrogen may play a role in protecting the kidney by decreasing oxidative stress and attenuating mechanisms linked to renal interstitial fibrosis.

Fig. 1.

Top: tomographic micro-computed tomography images of the renal microcirculation in young female controls, aged shams, and aged ovariectomized mice without (Ovx) or with estrogen replacement (Ovx+E). Bottom: quantitation of microvascular density showed that density of small renal cortical microvessels (<200 μm in diameter) was decreased in all aged groups, but most significantly in shams. The density of larger microvessels (200–500 μm) was decreased only in aged shams. Values are means ± SE. *P ≤ 0.05 vs. young control. #P ≤ 0.05 vs. Ovx+E.

Fig. 2.

Renal protein expression (all relative to GAPDH) of pro- and antiangiogenic factors, vascular endothelial growth factor (VEGF), VEGF receptors FLK-1 and FLT-1, basic-fibroblast growth factor (b-FGF), angiomodulin, angiostatin, and Notch-1 in young controls, aged shams, and aged Ovx or Ovx+E mice. Top left: representative bands. Top right and bottom: quantitation showing attenuated proangiogenic signaling in sham and attenuated antiangiogenic signaling in Ovx. The bar graph for b-FGF shows the 23-kDa form, and the 21-kDa form showed a similar pattern. Values are means ± SE. *P < 0.05 vs. young control.

Fig. 3.

Renal protein expression (all relative to GAPDH) of the profibrogenic factors transforming growth factor (TGF)-β and its mediator phospho-Smad3, plasminogen activator inhibitor (PAI)-1, endothelin (ET)-1, the angiotensin II type 1 (AT₁) receptor, protein kinase C (PKC), and nitrotyrosine in young controls, aged shams, and aged Ovx or Ovx+E mice. Top left: representative bands. Top right and bottom: quantitation showing increased profibrogenic signaling in Ovx, which was attenuated by estrogen replacement, as was the expression of nitrotyrosine, the footprint of peroxynitrite, and superoxide presence. Values are means ± SE. *P < 0.05 vs. young control. #P < 0.05 vs. Ovx+E.

Fig. 4.

Representative staining (blue, ×20; left) and quantitation (right) of renal trichrome (top) and dihydroethidium (DHE; bottom) in young controls (A), aged shams (B), and aged Ovx (C) or Ovx+E (D) mice. Renal fibrosis and oxidative stress in Ovx decreased after estrogen. Values are means ± SE. *P ≤ 0.05 vs. young control. #P < 0.05 vs. Ovx+E. †P < 0.05 vs. aged sham.

Fig. 5.

A diagram showing the pathways stipulated to be involved in the effects of aging and Ovx on the female mouse kidney. Aging impaired expressions of growth factors (VEGF, b-FGF), leading to renal microvascular rarefaction. Ovx induced oxidative stress and upregulated fibrogenic factors that subsequently induced renal fibrosis. Aging and Ovx seem to have dual effects on TGF-β expression, depending on the model and experimental conditions, as indicated by the parallel stimulatory (solid arrow) and inhibitory (dashed line) symbols. Estrogen replacement attenuated renal fibrosis by ameliorating renal oxidative stress and fibrosis in aged mice, but had minimal effects on angiogenesis.