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ORIGINAL RESEARCH

Quantification of Kidney Fibrosis Using Ultrasonic Shear Wave Elastography Experimental Study With a Rabbit Model Sung Kyoung Moon, MD, Sang Yoon Kim, MD, Jeong Yeon Cho, MD, Seung Hyup Kim, MD

Objectives—The purpose of this study was to evaluate the feasibility of ultrasonic shear wave elastography for quantification of renal fibrosis in an experimental rabbit model. Methods—Thirty-eight kidneys of 19 rabbits were studied and categorized into 3 groups: group I, ureter obstruction (n = 9); group II, renal vein occlusion (n = 10); and group III, normal control (n = 19). Before surgery, we measured stiffness at the renal cortex using shear wave elastography and evaluated the sonographic findings, including size, echogenicity, and resistive index. We repeated the same sonographic examinations weekly until the fourth week. The degree of histologically quantified fibrosis and the measured stiffness values were statistically compared. Results—There was no significant difference in the mean stiffness values for the renal cortex in the 3 groups before surgery (8.95 kPa in group I, 9.06 kPa in group II, and 9.74 kPa in group III; P > .05). However, the mean stiffness in each group on the last sonographic examination was significantly different (10.91 kPa in group I, 13.92 kPa in group II, and 9.77 kPa in group III; P = .003). Pathologically, the degree of fibrosis was also significantly different (3.62% in group I, 11.70% in group II, and 0.70% in group III; P < .001). The fibrosis degree and stiffness were positively correlated (ρ = 0.568; P = 0.01). Conclusions—Tissue stiffness measured by ultrasonic shear wave elastography was positively correlated with histopathologic renal fibrosis. Ultrasonic shear wave elastography may be used as a noninvasive tool for predicting renal fibrosis. Received November 26, 2013, from the Department of Radiology, Kyung Hee University Hospital, College of Medicine, Kyung Hee University, Seoul, Korea (S.K.M.); and Department of Radiology, Seoul National University Hospital, Seoul, Korea (S.Y.K., J.Y.C., S.H.K.). Revision requested January 7, 2014. Revised manuscript accepted for publication July 31, 2014. This study was supported by the Seoul National University Hospital Research Fund (grant 04-2010-0840). Address correspondence to Jeong Yeon Cho, MD, Department of Radiology, Seoul National University College of Medicine, 28 Yeongon-dong, Jongno-gu, Seoul 110-744, Korea. E-mail: [email protected] Abbreviations

ANOVA, analysis of variance; RI, resistive index doi:10.7863/ultra.34.5.869

Key Words—elastography; genitourinary ultrasound; kidney; shear wave; stiffness; tubulointerstitial fibrosis

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unctional impairment of the kidney is more affected by the degree of tubular interstitial damage than glomerular damage.1,2 Chronic hypoxia is known to be a final common pathway, which induces fibrogenesis in the renal tubulointerstitium. There are several causes of chronic renal failure induced by chronic hypoxia or ischemia, including glomerular disease. Until now, renal biopsy has been the reference standard method for diagnosis of the glomerular disease status. It is a safe procedure, and complications are rare, but it is invasive, and associated sampling errors have been reported.3 Therefore, a noninvasive and repeatable method is needed for monitoring renal interstitial fibrosis. Ultrasound elastography measures the tissue stiffness, viscosity, or elasticity4–6 and can be considered comparable to “palpation” for estimating tissue hardness. Static elastography, which is the pioneering elastographic technique, actually palpates the tissue by manual compression and estimates tissue hardness by comparing

©2015 by the American Institute of Ultrasound in Medicine | J Ultrasound Med 2015; 34:869–877 | 0278-4297 | www.aium.org

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Moon et al—Quantification of Kidney Fibrosis Using Ultrasonic Shear Wave Elastography

the ultrasonic radiofrequency signal obtained before and after compression.7 This technique has been generally applied to tumor imaging of the palpable organs such as the prostate, breast, and thyroid.8 However, it has some limitations such as operator dependence, reproducibility, and inapplicability to the internal organs such as the liver and kidneys. Consequently, a different elastographic approach was devised to enable virtual palpation of internal organs and tissues.9,10 The core technique of this kind of elastography is measurement of the velocity of shear waves propagating in the target tissue; shear waves propagate faster in harder tissues than in softer tissues. This technology has advanced continuously, and ultrasound and magnetic resonance elastography are currently the most widely used elastographic techniques.11,12 They are noninvasive and reproducible for quantification of tissue stiffness or fibrosis. There have been many studies on the usefulness of ultrasound and magnetic resonance elastography for assessment of hepatic fibrosis in patients with chronic liver disease.8,13–18 Recent clinical studies used noninvasive diagnostic tools (eg, acoustic radiation force impulse imaging and transient elastography) for transplanted kidney monitoring.19–21 These studies showed the clinical feasibility of ultrasound elastography for kidney evaluation. The purpose of this study was to demonstrate the feasibility of ultrasonic shear wave elastography for assessment of renal tubulointerstitial fibrosis and to validate its accuracy for quantification of renal fibrosis in an experimental rabbit model. We used the histopathologic degree of renal interstitial fibrosis as a reference standard.

Materials and Methods Rabbit Models The Institutional Review Board at our hospital approved this study. Forty kidneys from 20 New Zealand White rabbits (body weight, 2.5–3.0 kg; age, 16–18 weeks) were initially analyzed in this study. The rabbits were divided into 3 groups as follows: group I, unilateral ureteral obstruction (n = 10); group II, unilateral renal vein occlusion (n = 10); and group III, normal controls (n = 19). Groups I and II consisted of 20 left kidneys, and group III consisted of 20 contralateral right kidneys from groups I and II to maintain kidney function and ensure survival of the rabbits. The rabbits were anesthetized by intramuscular administration of 5-mg/kg zolazepam/tiletamine (Zoletil; Virbac Korea, Seoul, Korea), and 1- to 3-mg/kg xylazine hydrochloride (Rompun; Bayer Korea, Seoul, Korea).

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The abdominal and flank skin of the rabbits was prepared by shaving the hair, sterilizing with a povidone-iodine solution, and covering with a surgical drape. A laparotomy was performed at the midline abdomen, and the left ureter or left renal vein was exposed, isolated, and ligated with a 3-0 silk suture. After complete ureter obstruction in 10 rabbits and vein occlusion in another 10 rabbits was achieved, the surgical wound was closed. All surgical procedures were conducted in an aseptic environment. Because 1 rabbit in group I died before the final end point (2 weeks) of the study, groups I and III ultimately consisted of 9 and 19 kidneys, respectively. Postoperative Sonographic Examination Protocol and Equipment One radiologist, who is an expert in uroradiology, performed the preoperative (baseline) sonographic examinations. Examinations were repeated on the first and third postoperative days and weekly until the second week after surgery in all group I rabbits and 9 of 19 group III rabbits. For all group II and 10 of 19 group III kidneys, examinations were repeated on the first and third postoperative days and weekly thereafter until the fourth week after surgery. The experiment in group I kidneys was discontinued 14 days after surgery because it was difficult to maintain hydronephrosis models for more than 2 or 3 weeks. For sonographic examinations, the rabbits were anesthetized and placed in the right and left lateral decubitus positions for each kidney examination. Shear wave elastography, conventional sonography, spectral Doppler sonography, and color Doppler sonography were performed together with an Aixplorer ultrasound scanner (SuperSonic Imagine, Aix-en-Provence, France) and a 50mm SuperLinear transducer (4–15 MHz, 8-MHz central frequency). A display setting of 75 kPa was used for shear wave elastography. The stiffness of the renal cortex was measured at least 5 times in different points of different images, and the mean value was calculated (Figure 1). Color mapping representing tissue stiffness was conducted in the elastographic mode, and the region of interest was adjusted to the target area (the renal cortex) in the kidney. Stiffness (Young modulus in kilopascals) was then measured by locating the Q-box in the randomly selected target area within the color-mapping region-of-interest box. Every Q-box was located in 5 different images. In the conventional sonographic examination, the default thyroid setting was used. The 2-dimensional gain of the entire image was adjusted and optimized by using auto time-gain compensation. In the conventional sonographic examinations, the kidney size (craniocaudal length) was measured. The

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presence of a change in cortical echogenicity and anatomic abnormalities were evaluated in each kidney. The cortical echogenicity of group I and II kidneys was compared to that of contralateral kidneys (group III). The echogenicity of both kidneys was subjectively compared in the dual mode at less than 27% of the 2-dimensional gain of the default setting. In the spectral Doppler examination, Doppler signals were generally obtained from the interlobar arteries of the upper, mid, and lower portions of the kidney, and the mean resistive index (RI) was calculated in each kidney. In group II, the presence of recanalized venous flow and perirenal collateral flow was assessed on color Doppler sonography. Histopathologic Quantification of Interstitial Fibrosis After the rabbits were euthanized, the kidneys were removed and stained with hematoxylin-eosin stain and Masson trichrome for microscopic examination. One renal pathologist reviewed the tissue slides and performed the quantitative analysis of renal interstitial fibrosis using an automatic image analyzer (QWin Pro; Leica Microsystems, Inc, Bannockburn, IL) and was blinded to the shear wave elastographic results. The analysis was performed by calculating the proportion of the pixels of fibrotic tissues, which were shown as blue tissues in the Masson trichrome stain. Figure 1. Measurement of renal cortical stiffness on ultrasonic shear wave elastography. The color of the region of interest represents tissue stiffness. The color scale is shown at the top right. Stiffness was measured by locating the round Q-box in the target area within the color-mapping ROI box. The stiffness of the renal cortex in this kidney was 8.34 kPa.

Statistical Analysis The following dichotomous variables from the conventional and color Doppler sonographic examinations were analyzed by the χ2 or Fisher exact test: echogenicity change, atrophy, anatomic abnormality, recanalized venous flow, and collateral flow. Continuous variables, (mean kidney size, mean RI, and mean stiffness at the final examination) were compared by 1-way analysis of variance (ANOVA) and multiple-comparison analysis. Two-tailed P < .05 was regarded as statistically significant. The correlation between the measured tissue stiffness by shear wave elastography and the histopathologic fibrosis degree was evaluated by Spearman correlation analysis. Statistical analyses were performed with SPSS version 17.0 software (IBM Corporation, Armonk, NY).

Results The mean sizes, RI values, and stiffness of 38 kidneys on preoperative sonographic examinations are displayed in Table 1. No kidney abnormality was found on conventional or color Doppler sonography. There was no significant difference in sizes, RI values, and renal cortical stiffness among the 3 groups. Figure 2 shows a scattergram of cortical stiffness measured in 38 kidneys and the mean cortical stiffness of each group. Table 2 summarizes the continuous data from the final sonographic examinations and the degree of renal interstitial fibrosis from the histopathologic examinations in each group. The mean stiffness in the 3 groups was significantly different (10.91 kPa in group I, 13.92 kPa in group II, and 9.77 kPa in group III; P = .003) on 1-way ANOVA. However, there was no significant difference in RI values among the groups. Pathologically, the degree of renal interstitial fibrosis was also significantly different among the groups (3.62% in group I, 11.70% in group II, and 0.70% in group III; P < .001) on 1-way ANOVA (Figure 3). The multiple-comparison analysis revealed that group II had a significantly higher renal cortical stiffness and more severe histopathologic fibrosis than groups I and III (Figure 4). Spearman correlation analysis revealed a significant correlation between the degree of renal interstitial

Table 1. Preoperative Baseline Values Parameter Size, cm RI Stiffness, kPa

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Group I (n = 9) Mean SD 3.43 0.52 8.95

0.14 0.04 1.38

Group II (n = 10) Mean SD 3.25 0.52 9.06

0.15 0.07 1.05

Group III (n = 19) Mean SD 3.45 0.52 9.74

0.21 0.07 1.48

P .083 .962 .257

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fibrosis and stiffness (ρ = 0.568; P < .001). Figure 5 shows the trends in stiffness changes for the groups. Group I showed a slight increase in stiffness, and group II showed the most fluctuation in stiffness. In group II, stiffness decreased immediately after surgery and then increased continuously until reaching a plateau during the second week. The stiffness of the kidneys in group III remained relatively constant compared to the other groups. Renal interstitial fibrosis was arbitrarily classified into grade 1 (10%) according to the degree of histopathologic fibrosis. As most kidneys showed minimal interstitial fibrosis, and the quantification of fibrosis alone was performed for the entire renal cortex, the Banff classification was not used for the analysis according to the fibrosis grade. The mean stiffness and degree of the fibrosis are shown in Table 3 and Figure 6.

Figure 2. Cortical stiffness measured in 38 kidneys.

The mean stiffness of the renal cortex increased with an increase in the fibrosis grade. However, grade 2 had a wider range of stiffness, which overlapped with those of grades 1 and 3. Although the hydrostatic pressure was not assessed separately, it might have been a confounding factor in group I kidneys, which were mostly grade 2 (9 of 12). There was significant difference in kidney sizes among the groups (Table 2). The mean kidney size in group I increased continuously with time (Figure 7). Meanwhile, that of group II was slightly enlarged after surgery but then decreased continuously such that the mean size on the last examination was significantly smaller than those of the other groups. Group III showed gradual enlargement after the first week, which was a result of compensation for the functional impairment of the contralateral kidney. All of the group I kidneys were found to have hydronephrosis without parenchymal atrophy and a considerable echogenicity change on conventional sonography. Two group I kidneys (22.2%) showed slightly increased renal cortical echogenicity compared to the contralateral kidneys. One kidney had renal parenchymal rupture and perirenal urinoma formation at the second-week examination. Six group II kidneys (60%) showed atrophic changes, and all group II kidneys (100%) showed increased cortical echogenicity (Figure 4), which appeared in the third week. Kidneys with increased cortical echogenicity (25 of 38 [65.8%]) had significantly higher mean stiffness (10.33 versus 12.66 kPa; P = .016), a more severe atrophic change (3.36 versus 3.87 cm; P = .010), and more severe histopathologic fibrosis (9.63% versus 1.51%; P < .001) than kidneys without a cortical echogenicity change (13 of 38 [65.8%]). Recanalized venous flow in the incompletely occluded main renal vein and perirenal collateral flow was seen in 9 kidneys (90%) and 8 kidneys (80%) in group II, respectively. In group III, only 1 kidney (0.05%), which was a contralateral kidney of group I, showed increased cortical echogenicity with a mean stiffness of 12.43 kPa and only 0.85% histopathologic fibrosis.

Table 2. Final Sonographic and Histopathologic Results Parameter

Group I (n = 9) Mean SD

Group II (n = 10) Mean SD

Size, cm RI Stiffness, kPa Histopathologic fibrosis, %

4.34 0.63 10.91 3.62

3.17 0.56 13.92 11.70

0.39 0.13 4.85 1.22

0.51 0.31 1.57 4.45

Group III (n = 19) Mean SD 3.67 0.61 9.77 0.70

0.23 0.14 1.25 0.26

P

Quantification of kidney fibrosis using ultrasonic shear wave elastography: experimental study with a rabbit model.

The purpose of this study was to evaluate the feasibility of ultrasonic shear wave elastography for quantification of renal fibrosis in an experimenta...
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