Fig 1.
Assessment of kidney weight, blood pressure, heart rate, locomotor activity, urinary output and urinary albumin excretion.
A) Kidney weights after 11 weeks of 2K1C. *p < 0.05. B) Weekly blood pressure measurements for 11 weeks after insertion of the renal artery clip. The difference in blood pressure between baseline and after clip placement is shown. *p < 0.001 vs. Control, **p < 0.0001 vs. Control. C) Circadian changes of blood pressure at baseline, 2 weeks and 11 weeks after clip placement. *p < 0.05 vs. light. D) Weekly heart rate measurements for 11 weeks of 2K1C. *p < 0.001 vs. baseline (0 weeks). E) Circadian changes of heart rate at baseline, 2 weeks and 11 weeks after clip placement. *p < 0.05 vs. light. F) Locomotor activity in mice over 11 weeks of 2K1C. G) Circadian changes of activity at baseline, 2 weeks and 11 weeks after clip placement. *p < 0.05, **p < 0.01, ***p < 0.001 vs. light. H) Urinary output at baseline and 1, 2, 3, 4, 6, 8, and 10 weeks after clip placement. I) Daily urinary albumin excretion at baseline and 1, 2, 3, 4, 6, 8, and 10 weeks after insertion of the renal artery clip *p < 0.0001 vs. Control.
Fig 2.
Histological analysis of kidney sections 2 and 11 weeks after renal artery clip placement.
A) PAS staining of control, 2K1C unclipped and 2K1C clipped kidneys 2 and 11 weeks after clip placement. B) Masson’s Trichrome staining of control, 2K1C unclipped, and 2K1C clipped kidneys 2 and 11 weeks after clip placement. *p < 0.001 vs. Control, p** < 0.001 vs. 2 wks, #p < 0.001, $p < 0.01.
Fig 3.
Expression of renal PRCP, PREP, ACE and renin activity 2 and 11 weeks after renal artery clip placement.
A) Western Blot of renal PRCP in control, 2K1C unclipped and 2K1C clipped groups after 2 weeks of 2K1C. B) Western Blot of renal PRCP in control, 2K1C unclipped and 2K1C clipped groups after 11 weeks of 2K1C. C) Western Blot of renal PREP in control, 2K1C unclipped, and 2K1C clipped groups after 11 weeks of 2K1C. D) Western Blot of renal ACE in control, 2K1C unclipped, and 2K1C clipped groups after 2 weeks of 2K1C. E) Western Blot of renal ACE in control, 2K1C unclipped, and 2K1C clipped groups after 11 weeks of 2K1C. F) Mass spectrometric analysis of renin activity in control, 2K1C unclipped, and 2K1C clipped groups. For Western blots, protein levels were normalized to β-actin. Values are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4.
PRCP enzyme activity in control, 2K1C unclipped and 2K1C clipped groups 2 and 11 weeks after clip placement.
A) MALDI imaging of Ang-(1–7) formation from Ang II in control kidneys or unclipped and clipped kidney sections obtained after 2 weeks of clip placement. *p < 0.01 vs. Control. B) In vitro enzyme activity of Ang-(1–7) formation using homogenates obtained from control kidneys or unclipped and clipped kidneys 11 weeks after renal artery clip placement. *p < 0.01 vs. Control. C) Plasma PRCP activity at baseline and 2 or 11 weeks after clip placement. D) PRCP activity in 24-hour urine samples at baseline and 1, 2, 3, 4, 6, 8, and 10 weeks after insertion of the renal artery clip.
Fig 5.
Immunofluorescence analysis of PRCP and nephrin at 60x magnification eleven weeks after 2K1C.
White arrows indicate PRCP staining. A) Localization of PRCP and nephrin with overlay in the control kidney. B) Localization of PRCP and nephrin with overlay in the unclipped kidney. C) Localization of PRCP and nephrin with overlay in the clipped kidney. D) Quantitative analysis of cortical, glomerular and tubular PRCP expression in control, unclipped and clipped kidneys. *p < 0.05 vs. Control. **p < 0.01 vs. Unclipped.