Novel Use of Ultrasound to Measure Regional Kidney Volume Changes Post Ischemia Reperfusion (IR) in Male and Female Rats

Abstract

Swelling is associated with injury following ischemia reperfusion (IR). The goal of this study was to test the efficacy of ultrasound (US) to quantify regional kidney volume in rats following IR. In order to determine whether US imaging can detect biologically relevant changes in renal volume, we measured renal cortical and medullary volumes in control male and female rats and 24 hours post IR and compared these data to the gold‐standards for measuring volume, stereology and fluid displacement for whole kidney. 13 week old male and female spontaneously hypertensive rats (SHR) were randomized to control or 45 minute warm bilateral ischemia followed by 24 hours of reperfusion (n=5–6). 3D US imaging (Vevo770) was used to measure left kidney volume prior to sacrifice. Following US imaging, the left renal hilus was ligated, the kidney excised and volume was determined by fluid displacement in a water filled conical tube. For stereological measurements of regional kidney volumes, the right kidney was pressure perfusion fixed (males @175mmHg; females @155mmHg), excised and placed in formalin for 72 hours. Kidneys were sliced into transverse sections of 1.075mm using a multi‐blade apparatus. A 0.846mm grid was placed over every other slice and kidney volume was estimated using the Cavalieri's Principle. IR significantly increased whole kidney volume in male (US: 881 to 994mm3; stereology: 1235 to 1343mm3) and female SHR (US: 611 to 786mm3; stereology: 776 to 824mm3) vs. controls (p<0.0001). Regional kidney volumes, as assessed by US, correlated with stereological measurements of cortical and medullary volume (medulla: p=0.0003; cortex: p=0.0002). The slopes of these relationships (medulla=0.6±0.1; cortex=0.6±0.1) as well as the y‐intercepts (medulla=23%±13%; cortex=5%±15%) indicate that relative to stereological measurements, US tended to overestimate the volume of small kidneys and underestimate volumes of large kidneys. In regards to sensitivity of the measurement, an increase in volume selectively in the renal medullary region was detected in both male (404 to 451mm3; p<0.0001) and female SHR (234 to 279mm3; p<0.0001) post IR when measured by stereology. When measured by US, however, both the medulla (male: 247 to 309mm3; female: 149 to 218mm3; p<0.0001) and cortex (male: 634 to 686mm3; female: 462 to 568mm3; p=0.03) were found to increase in volume following IR. Our data also indicate that whole kidney volume measurements obtained via US are in good agreement with fluid displacement (p<0.0001) and stereology (p<0.0001) with the skewness of the regression line for stereology matching the analysis of the regional comparisons. In conclusion, our studies validate the use of 3D US imaging for determining biologically relevant changes in regional kidney volumes in rats. Furthermore, our data suggests US may be more sensitive in determining these changes compared to stereology. US imaging is advantageous as it provides a non‐invasive way of determining relative regional kidney volumes over time. Interestingly, both methods indicate an increase in medullary volume in response to IR in male and female SHR. Studies are needed to better define the contribution of the medulla to IR injury.