In Vivo Renin Activity Imaging in the Kidney of Progeroid Ercc1 Mutant Mice

Figures

• 

  Figure 1. Histopathological changes in the kidneys of progeroid Ercc1d/− mice. Representative pictures of hematoxylin and eosin (HE) (a), and periodic acid–Schiff (PAS) staining (b) of the kidneys of 6- and 24-week-old Ercc1d/− mice, their wild-type (WT) littermates and old WT mice (104 weeks of age). Histological examination showed signs of kidney aging, including anisokaryosis, tubular degeneration, loss of brush borders and glomerulosclerosis in kidneys of Ercc1d/− mice at 24 weeks of age (indicated by * in the PAS staining). In all panels, scale bar = 100 μm.

• 

  Figure 2. Functional renal changes in progeroid Ercc1d/− mice. (a) The number of glomeruli confirmed normal kidney development from birth in Ercc1d/−. (b) ATN scoring showed significantly increased kidney damage in 24-week-old Ercc1d/− mice compared to their wild-type (WT) littermates. (c). Quantification of TUNEL staining indicated significantly increased apoptotic cell death in Ercc1d/− kidneys. Urinary albumin (d), creatinine (e) and urea (f) levels were comparable between 24-week-old Ercc1d/− and WT mice. (g) Urea/creatinine ratio showed no significant differences between Ercc1d/− and WT mice. (h) Plasma renin concentration was significantly decreased in 24-week-old Ercc1d/− mice compared to WT. Data are means ± SEM of n = 4–6 (a–c) and n = 6–14 (d–h). Note: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT.

• 

  Figure 3. Specific in vitro enzymatic activation of ReninSense by kidney and plasma renin. (a) ReninSense was rapidly activated by kidney renin in WT mice. Low levels of fluorescence were found in RenKO kidney lysates comparable to the autofluorescence of the probe. High concentrations of aliskiren completely blocked ReninSense activation. (b) The half-maximal inhibitory concentration (IC50) for aliskiren in kidney lysates was 10−7.7 M. Data are means ± SEM of duplicate samples. Note: *** p < 0.001 vs. control.

• 

  Figure 4. In vivo activation of ReninSense in kidneys of WT mice, with and without losartan treatment. (a) Mice were imaged tomographically by FMT 2500 and microCT 24 h after ReninSense injection. Micro CT imaging and FMT imaging of Annexin-Vivo allowed accurate localization of the kidneys. Combined microCT and FMT imaging of Annexin-Vivo and ReninSense showed in vivo renin activity in the kidneys and bladder (clearance of probe). (b) Losartan-treated mice showed increased in vivo intrarenal renin activity, which was confirmed by quantification. (c,d) Ex vivo imaging of the kidneys by the Odyssey® system confirmed activation of the ReninSense probe in losartan-treated mice. Fluorescence of ReninSense could not be detected in vivo or ex vivo in RenKO mice. (e) Losartan treatment increased plasma renin activity. (f) Increased expression levels of renin in the kidneys were found in losartan-treated mice. ND, not detectable. Data are means ± SEM of n = 3. Note: * p < 0.05, *** p < 0.01 vs. WT.

• 

  Figure 5. In vivo imaging of renin activity by ReninSense in progeroid Ercc1d/− mice. (a) Ercc1d/− mice display activated intrarenal renin activity at 24 weeks of age, as evidenced by increased fluorescence detected with the ReninSense probe when imaged with the microCT and FMT. (b) Quantification of the in vivo fluorescence of ReninSense confirmed increased renin activity in the kidneys, which was significantly different at 24 weeks of age. (c) These results were further confirmed ex vivo by imaging of the 24-week-old kidneys with the Odyssey® system. Data are means ± SEM of n = 5–15 (b) and n = 3–12 (d). Differences were assessed by one-way ANOVA, followed by correction for multiple testing by post hoc Bonferroni analysis. Note: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT.