Novel use of ultrasound to examine regional blood flow in the mouse kidney

Abstract

Conventional methods used for measuring regional renal blood flow, such as laser-Doppler flowmetry, are highly invasive, and each measurement is restricted to a discrete location. The aim of this study was to determine whether ultrasound imaging in conjunction with enhanced contrast agent (microbubbles; Vevo MicroMarker, VisualSonics) could provide a viable noninvasive alternative. This was achieved by determining changes in renal cortical and medullary rate of perfusion in response to a bolus injection of endothelin-1 (ET-1; 0.6, 1.0, or 2.0 nmol/kg) and comparing these responses to those observed in separate groups of mice with conventional laser-Doppler methods. Intravenous infusion of ET-1 in anesthetized male C57bl/6 mice resulted in a dose-dependent increase in mean arterial pressure and a dose-dependent decrease in total renal blood flow as measured by pulse-wave Doppler. ET-1 infusion resulted in a dose-dependent decrease in regional kidney perfusion as measured by both ultrasound with enhanced contrast agent and laser-Doppler measurements, verifying the use of ultrasound to measure regional kidney perfusion. Noted limitations of ultrasound imaging compared with laser-Doppler flowmetry included a lower degree of sensitivity to changes in tissue perfusion and the inability to assess rapid or transient changes in tissue perfusion. In conclusion, ultrasound represents an effective and noninvasive method for the measurement of relatively short-term, steady-state changes in regional blood flow in the mouse kidney.

Fig. 1.

Representative ultrasound images of a mouse kidney. Top left: reference image of a mouse kidney obtained using contrast mode with an overlay to indicate renal anatomy. Top right: gray-scale image in contrast-mode following the infusion of a contrast agent. The green color indicates the presence of microbubbles in the renal circulation. Bottom left: reconstructed contrast-mode image under baseline conditions derived from subtraction of top left from top right and showing only microbubbles. Bottom right: reconstructed contrast-mode image 20 min after the infusion of endothelin (ET)-1. Note the reduced intensity of microbubbles compared with bottom left, indicating reduced renal perfusion following ET-1 infusion. IM, inner medulla; OM, outer medulla.

Fig. 2.

Representative time-intensity curves in a region of interest in contrast mode. A: representative time-intensity curve with curve fit under baseline conditions. B: representative time-intensity curve with curve fit 2 min post-ET-1 infusion. Note that at 2 min after the infusion of ET-1, there is not a measureable increase in contrast agent influx, making measurements at this time point unusable.

Fig. 3.

Percent change in mean arterial pressure (A) and heart rate (B) 20 min after the infusion of saline or ET. #Significant difference from saline infusion, P < 0.01; n = 6.

Fig. 4.

Total renal blood flow velocity measured using pulse-wave Doppler (PW-mode) in the renal artery. A: representative image depicting the location from which measurements were made. B: averages; n = 6.

Fig. 5.

Analysis of renal hemodynamic properties of the renal cortex in response to bolus infusion of saline or ET-1 using ultrasound in conjunction with a contrast agent. Cortical perfusion or blood volume is determined by contrast agent intensity/mm² in the region of interest (A and B). Blood velocity (C) was determined using the following equation: contrast intensity = B + A[1 − exp(−βt)], where B is reference intensity, A is plateau signal intensity, the exponential rate parameter β is the velocity of the microbubbles entering the imaging plane, and t is a constant. Tissue perfusion (D) is calculated by (A − B) * β. *Significant difference from saline infusion, P < 0.05. +Difference from saline infusion, P = 0.07; n = 6–9.

Fig. 6.

Analysis of renal hemodynamic properties of the renal medulla in response to bolus infusion of saline or ET-1 using ultrasound in conjunction with contrast agent. Outer medullary perfusion or blood volume is determined by contrast agent intensity/mm² in the region of interest (A and B). Blood velocity (C) was determined using the same equation as for Fig. 5 as was tissue perfusion (D). *Significant difference from saline infusion, P < 0.05; n = 7–9.

Fig. 7.

Examination of the day-to-day (A and B) and mouse-to-mouse (C and D) variation in the rate of tissue perfusion assessed by ultrasound. Tissue perfusion was measured in the renal cortex and medulla in response to a bolus infusion of ET-1 (2 nmol/kg). There were no significant differences; n = 5.

Fig. 8.

Cortical (A; n = 5–7) and medullary (B; n = 4–7) perfusion measured as assessed by red blood cell flux by laser-Doppler 20 min after the infusion of saline or ET. *Significant difference from saline infusion, P < 0.05. #Significant difference from saline infusion, P < 0.01.