Med Mol Morphol DOI 10.1007/s00795-017-0157-8

ORIGINAL PAPER

Quantified kidney echogenicity in mice with renal ischemia reperfusion injury: evaluation as a noninvasive biomarker of acute kidney injury Shinya Murata1,2 · Noriyuki Sugiyama1 · Kentaro Maemura1 · Yoshinori Otsuki3 

Received: 29 January 2017 / Accepted: 11 March 2017 © The Japanese Society for Clinical Molecular Morphology 2017

Abstract  The purpose is to evaluate quantified kidney echogenicity as a biomarker for the early diagnosis of acute kidney injury (AKI) and predicting progression to chronic kidney disease (CKD) in a mouse model of ischemia–reperfusion injury (IRI). Two separate protocols of murine models of IRI were used: (1) 10, 30, and 40 min of bilateral ischemia duration and (2) 45 and 60 min of unilateral ischemia duration. Renal echogenicity was measured with ultrasound and compared with serum creatinine or urine neutrophil gelatinase-associated lipocalin (NGAL) at various timepoints after IRI. In mice subjected to 10, 30, and 40  min of bilateral ischemia, renal echogenicity increased about 2  h after IRI for all ischemia times, earlier than serum creatinine or urine NGAL. In those subjected to 45 and 60 min of unilateral ischemia, 60 min of unilateral ischemia, which represents atrophic changes 28 days after IRI, resulted in a sustained high level of echogenicity and was significantly different 24 h after IRI, while 45 min of * Noriyuki Sugiyama an1027@osaka‑med.ac.jp Shinya Murata [email protected] Kentaro Maemura an2011@osaka‑med.ac.jp Yoshinori Otsuki y.otsuki@osaka‑med.ac.jp 1

Division of Life Science, Department of Anatomy and Cell Biology, Osaka Medical College, 2‑7 Daigaku‑machi, Takatsuki, Osaka 569‑8686, Japan

2

Department of Pediatrics, Hirakata City Hospital, 2‑14‑1 Kinyahommachi, Hiralata, Osaka 573‑1013, Japan

3

Osaka Medical College, 2‑7 Daigaku‑machi, Takatsuki, Osaka 569‑8686, Japan





unilateral ischemia resulted in trivial levels of histological damage 28  days after IRI. Renal echogenicity might have the potential to be a biomarker for the early diagnosis of AKI and the prognosis of CKD. Keywords  Acute kidney injury · Biomarker · Chronic kidney disease · Echogenicity · Ultrasound

Introduction Acute kidney injury (AKI) is defined as a clinical condition in which renal function deteriorates rapidly within 48 h [1]. AKI remains a significant clinical problem because of its high mortality and morbidity rates [2]. Moreover, recent studies have reported that a significant proportion of patients with AKI develop a predisposition toward chronic kidney disease (CKD), which is characterized by a chronic decrease in renal function and renal atrophy resulting from irreversible histological damage [1]. Although the primary indicator of renal function has typically been serum creatinine and urine volume, significant increases in serum creatinine are observed 24–48  h after the renal injury event, and cases in which early onset AKI present with oliguria are rare. Thus, investigation of therapeutic interventions for AKI and prognostic indicators of CKD, beyond simply monitoring serum creatinine or urine volume, is required. Therefore, the search for sensitive biomarkers of AKI enabling early therapeutic intervention and prediction of disease prognosis is currently ongoing. Sensitive biomarkers such as neutrophil gelatinase-associated lipocalin (NGAL) have been reported [3–5]. However, they have not entered routine clinical use. It has long been known that renal echogenicity increases in AKI [6–9]. Ultrasound examination is less invasive than blood and urine sampling, and frequent

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measurements can be performed over time. However, no reports have evaluated renal echogenicity over time as a biomarker of AKI from the early stages of a renal injury event. The aim of this study was to evaluate renal echogenicity as a noninvasive biomarker of early AKI diagnosis and as a predictor of histological damage in a mouse model of ischemia reperfusion injury (IRI), in comparison with previously reported biomarkers, such as serum creatinine and urine NGAL.

Materials and methods Mouse models of renal IRI All studies were approved by the Committee for Animal Research, Osaka Medical College. We used well-established murine models of renal IRI [10]. FVB/N male mice, aged 4–8 weeks and weighing 22–26  g, were used. Two separate protocols of renal IRI were used: (1) 10, 30, and 40  min of bilateral ischemia (to establish a mouse model of early AKI) and (2) 45 and 60 min of unilateral ischemia (to establish a mouse model of AKI-CKD) [11]. The number of experimental animals in each group including the control (Sham) group was five. The IRI surgery was done under anesthesia with an intraperitoneal injection of 50 mg/ kg pentobarbital sodium and the mice were placed on a homeothermic table to maintain their body temperature at 40 °C. Flank incisions were made to expose the renal pedicles. The renal pedicles were clamped unilaterally or bilaterally with arterial microclamps. The clamp was then removed after each ischemic episode, and the kidney was observed for the return of blood flow. Urine was collected before IRI, and after 2, 6, 12, and 24  h of reperfusion. Urine NGAL levels were measured using a commercially available enzyme-linked immunosorbent assay [ELISA] kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. Blood sampling was performed at the same time via puncture of the inferior vena cava (the number of this experimental animals in each time including before IRI was five) for measurement of serum creatinine (LSI Medience Co., Tokyo, Japan). Sham control mice were subjected to identical surgery except for the renal pedicle clamping. Ultrasound measurement of renal echogenicity The Aplio 300 ultrasound system (Toshiba Medical Systems, Otawara, Tochigi, Japan) with a linear ultrasound probe (PLT-1202s) at a frequency of 13  MHz was used. Mice were anesthetized, and the left dorsal area was shaved prior to measuring the echogenicity of the left kidney and spleen at various points. Echogenicity of

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the kidney and spleen was quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). In light of the individual differences in ultrasound examination conditions, renal echogenicity was determined as a ratio in reference to that of the spleen. The region of interest (ROI) measurements of the kidney were obtained from the entire left kidney in the longitudinal plane (setting the renal hilum and renal pelvis as landmarks), because corticomedullary differentiation was diminished especially in injured kidney. The ROI measurements of the spleen were obtained from the spleen observed in the same field of view. Three measurements of the mean intensity were performed on each image, and the average value was calculated. The ultrasound examination was done under anesthesia with 3.0% sevoflurane plus air and oxygen (­FiO2 0.5) in an acrylic glass chamber. The echogenicity measurements were made in the mice with 10, 30, and 40 min of bilateral ischemia before IRI, and 2, 6, 12, and 24  h after IRI. The echogenicity measurements were made in the mice with 45 and 60 min of unilateral ischemia before IRI, and 2 h, 6 h, 12 h, 24 h, 7 days, 14 days, and 28 days after IRI. Histological analysis Kidneys were fixed in 4% paraformaldehyde for paraffin embedding. Paraffin-embedded tissues were sectioned at a thickness of 4  μm along the long axis at the center of the kidney. Five kidneys in each group were examined in all histological examinations. Sections were stained with PAS (Sigma-Aldrich Japan, Tokyo, Japan) and Masson trichrome (Applied Medical Research Laboratory, Osaka, Japan). The degree of renal injury was evaluated using PAS-stained sections. For semi-quantitative analysis, the extent of tubule injury (determined by cell lysis, loss of brush border, and cell detachment) was scored as follows: 0: 0–25%; 1: 25–50%; 2: 50–75%; and 3: 75–100%. We have described this as the “Kidney Injury Score” [11–14]. Interstitial fibrosis was evaluated using Masson trichrome-stained sections with Adobe Photoshop Version 5.5. The histological analysis was done using 10 randomly selected fields per section using a random number table. To establish the mouse model of early AKI Serum creatinine and histological damage in the kidney were evaluated after 10, 30, and 40  min of bilateral ischemia at 2, 6, 12, and 24  h. The early AKI model was defined as a significant increase of serum creatinine and the “Kidney Injury Score”.

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To establish the mouse model of AKI‑CKD

Statistical analysis

Kidney weight / body weight (KW/BW) was evaluated after 45 and 60  min of unilateral ischemia at 1, 7, 14, and 28 days (The number of this experimental animals in each time including before IRI was five). The AKI-CKD model was defined as significant renal atrophy that was the same as the significant decrease of kidney weight / body weight (KW/BW) 28  days after IRI (versus Sham) [12, 15].

Data are expressed as means ± standard deviation (SD). The vertical bars of the graphs are error bars that represent SD. Comparisons between groups were performed using a nonparametric test (Wilcoxon test), with P < 0.05 considered significant. Statistical analysis was performed using the JMP® 12 software (SAS Institute Inc., Cary, NC, USA).

Results Characterization of the early AKI mouse model

Fig. 1  Bar graph showing that serum creatinine is significantly elevated after 30 (B30) and 40 (B40) min of bilateral ischemia 24 h after reperfusion (*P < 0.05 versus Sham, †P < 0.05 versus 10 min of bilateral ischemia (B10), §P < 0.05 versus B30). The vertical bars of the graphs are error bars showing standard deviation

Serum creatinine levels were significantly elevated in the 24  h reperfusion samples after 30 and 40  min of bilateral ischemia, compared with Sham and 10  min of bilateral ischemia (P < 0.05). Serum creatinine was increased significantly more with 40 min of bilateral ischemia than with 30  min of bilateral ischemia (Fig.  1). Characteristic histopathologic features of ischemic injury, i.e., tubular cell lysis and detachment [4, 16], were evident in the 24 h reperfusion samples after 30 and 40 min of bilateral ischemia, whereas histologic changes were minimal after 10  min of bilateral ischemia (Fig.  2a–d). Kidney injury scores were significantly elevated in the 24 h reperfusion samples after 30 and 40 min of bilateral ischemia (P < 0.05 versus Sham

Fig. 2  Photomicrographs (bar 100  μm) show representative histological kidney samples stained with PAS at 24  h after a 0  min of ischemia (Sham), b 10  min of ischemia, c 30  min of ischemia, and d 40  min of ischemia. Histological injury, i.e., loss of brush border or cell detachment of the tubular epithelium, is observed in all ischemia groups (10, 30, and 40  min). e Kidney injury score 24  h

after ischemia–reperfusion injury. The score is significantly elevated following 30 (B30) and 40 (B40) min of bilateral ischemia (*P < 0.05 versus Sham, †P < 0.05 versus 10  min of bilateral ischemia (B10), § P < 0.05 versus B30). Histological injury is proportional to ischemia duration. The vertical bars of the graphs are error bars showing standard deviation

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and 10 min bilateral ischemia) (Fig. 2e). Mice subjected to 10 and 30 min of bilateral ischemia all survived 24 h after IRI, but mice subjected to 40  min of bilateral ischemia showed high mortality (50%; 5/10 dead 24 h after IRI).

(Fig. 4d). Although renal areas in 10 min ischemia samples were decreased slightly after 12 h, there was no significant difference in the size of the renal area of the ischemia samples at 30 and 40 min.

Renal echogenicity after renal ischemia–reperfusion in the early AKI model

Characterization of the AKI‑CKD mouse model

First, echogenicity was monitored for up to 24 h after renal IRI in 30-min bilateral ischemia mice. The echogenicity of the renal parenchyma was elevated within 2  h after renal IRI was then confirmed (Fig. 3). Second, echogenicity was quantified with each ischemia episode. The echogenicity of the 10, 30, and 40 min bilateral ischemia samples was significantly elevated at 2 h after IRI. The echogenicity of the 10 min of ischemia group returned to levels similar to that of Sham (and before IRI) at 6  h. The echogenicity of the 30 min of ischemia group returned to levels similar to that of Sham (and before IRI) at 24 h. The echogenicity of the 30 and 40 min of ischemia groups remained elevated at 6 h post-IRI, while the echogenicity of the 40 min of ischemia group remained elevated at 24 h post-IRI (Fig. 4a). Third, these results were compared with previously reported biomarkers of AKI, serum creatinine and urine NGAL (Fig.  4b, c). After 30 and 40  min of ischemia, serum creatinine began to increase at 6 h after IRI, and it increased until 24 h. All ischemic durations produced increased urine NGAL at 6  h after IRI, which continued to increase until 24 h. Notably, the results showed that the echogenicity for all ischemia durations was significantly elevated 2  h after IRI, earlier than serum creatinine and urine NGAL. Entire renal areas in 30 and 40  min bilateral ischemia samples were expanded compared to before ischemia

Fig. 3  Region of interest (ROI) measurements of the kidney are obtained from the entire left kidney in the longitudinal plane (blue line). The ROI measurements of the spleen are obtained from the spleen observed in the same field of view (yellow line). Ultra-

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Kidney weight /  body weight (KW/BW) in the 60  min of ischemia group decreased beginning 14 days after IRI, and it was significantly lower 28 days after IRI compared with the 45  min of ischemia group (Fig.  5a). Kidney appearance in the 60  min of ischemia group 28 days after IRI showed remarkable atrophic change (Fig. 5b). While histological examination at 28  days after IRI revealed that the tubular structure was almost restored and the area of interstitial fibrosis was not increased with 45  min of ischemia (Fig. 5c), whereas after 60 min of ischemia, loss of tubular structure and increased interstitial fibrosis were observed (Fig. 5d, e). Renal echogenicity after renal IRI in the AKI‑CKD model Echogenicity was monitored for up to 28 days after IRI in mice subjected to 45 and 60  min of unilateral ischemia. The echogenicity of the renal parenchyma was maintained at a high level and showed atrophic changes 28 days after 60  min of ischemia compared to the 45  min of ischemia group (Fig.  6a, b). The renal echogenicity of both the 45 and 60 min of unilateral ischemia began to decrease around 24  h after IRI. Furthermore, the renal echogenicity of 45  min of unilateral ischemia recovered to a level almost

sound images before and after IRI (2, 6, 12, and 24  h) in the Sham group and following 30 min of bilateral ischemia. The images show that increased renal echogenicity begins around 2  h post-IRI and decreases around 6–12 h post-IRI. (Color figure online)

Med Mol Morphol Fig. 4  Graphs show a echogenicity, b serum creatinine, c urine NGAL, and d kidney size at multiple timepoints after bilateral ischemia [10 min (red line), 30 min (green line), and 40 min (purple line)] [*P < 0.05 versus Sham (blue line) at the same timepoint, †P < 0.05 versus pre-IRI for each ischemia duration]. The vertical bars of the graphs are error bars showing standard deviation. (Color figure online)

equal to Sham around 28 days, whereas 60 min of ischemia maintained a high level of echogenicity (Fig. 6c).

Discussion In this study, mice subjected to 30 and 40 min of bilateral ischemia were deemed to be an early AKI model, with a significant increase of serum creatinine and histological injury, while those subjected to 10 min of bilateral ischemia were deemed to be a subclinical AKI model; those subjected to 60 min of unilateral ischemia were deemed to be an AKI-CKD model (atrophic model), which represented significant renal atrophy, while those subjected to 45  min of unilateral ischemia were deemed to be a repaired AKI model. Using these mice models, it was shown that renal echogenicity might have the potential to be a biomarker for the early diagnosis of AKI and the prognosis of CKD. There are a number of reports about AKI biomarkers, such as NGAL [17], L-type fatty acid-binding protein (L-FABP) [18–20], kidney injury molecule-1 (KIM-1) [21, 22], and interleukin-18 (IL-18) [23]. Increases in urine NGAL, urine L-FABP, KIM-1, and IL-18 are observed within 2–6, 4, 6–24, and 4–6  h after renal injury, respectively, in humans. However, frequent measurement of these biomarkers is invasive because of the requirement for serum and urine samples. Ultrasound is a noninvasive method that allows for frequent measurements in real time. Renal ultrasonography has been commonly used to evaluate renal function.

The features of AKI in ultrasonography are (1) enlargement of kidney size, (2) increased resistive index (RI), and (3) increased echogenicity. First, regarding kidney size, Nomura et  al. reported that the ratio of anteroposterior to longitudinal diameter was correlated with serum creatinine [8]. However, the present results showed no significant difference in KW/BW among various ischemia durations within 24 h after IRI. By ultrasonography, the size of kidney was expanded by ischemia more than 30 min, but the relationship between ischemic time and kidney size was not observed. Therefore, we suggest that kidney size might not be suited for the AKI diagnosis. Second, RI is determined by assessing systolic and diastolic blood velocities in the segmental arteries and applying the following formula: (peak systolic velocity - end diastolic velocity)/peak systolic velocity. RI is an indicator of resistance to flow in the kidney, which has been said to be correlated with renal function and reproducible when performed by a trained sonographer [24, 25]. For accurate evaluation over time, RI should be calculated with the velocities obtained from the same transducer angle [7]. If the subject is small and the respiration motion is large or frequent, it is difficult to track vessels and measure RI. For that reason, it was not possible to measure and evaluate RI in the current study using mice. Third, echogenicity is easily measured, even by general physicians and medical technologists. Some reports have suggested that the degree of increase in renal echogenicity might reflect the severity of renal disease [26–28]. However, correlations among renal echogenicity, renal function, and histopathological change for early onset of

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Fig. 5  Graph a shows the time course of kidney weight / body weight (KW/BW) of Sham (empty circle), 45  min (filled square), and 60  min (empty square) of unilateral ischemia (*P < 0.05 versus Sham at the same timepoint, †P < 0.05 versus 45  min of unilateral ischemia at the same timepoint). Photograph b shows the appearance of kidneys exposed to 45  min (left) and 60  min (right) of unilateral ischemia 28  days post-IRI (bar 10  mm). Photomicrographs

show histological change at 28 days after 45 min (c) and 60 min (d) of unilateral ischemia, as determined by Masson trichrome staining (bar 100  μm). e Bar graph shows that the fibrotic index is significantly elevated following 60  min of unilateral ischemia 28 days post-IRI (*P < 0.05 versus Sham, †P < 0.05 versus 45  min of unilateral ischemia). The vertical bars of the graphs are error bars showing standard deviation

AKI were not evaluated. The current study addressed this point. The present results indicated that renal echogenicity could be correlated with renal function and histopathological changes in the early phase of AKI. Our results indicated that the echogenicity in AKI-CKD kidney was significantly higher than repaired AKI kidney from 1 to 28  days after IRI. This increase in echogenicity is thought to be due to acute and chronic histological damages. Although the acute damage of making high echogenicity up to day 7 after IRI was unknown, the interstitial fibrosis is likely cause of the chronic damage of making high echogenicity. Interstitial fibrosis began from day 14 after unilateral IRI and the fibrotic areas were ubiquitously spread. It has been known that RI by ultrasonography was correlated with the pathological changes including glomerulosclerosis, arteriosclerosis, and interstitial fibrosis in the CKD patients [29]. Likewise, renal echogenicity might reveal the same pathological change. Regarding the quantified renal echogenicity to measure, absolute echogenicity has large variations in individuals,

because there are some organs such as connective tissue between the body surface and kidneys that can influence echogenicity. Thus, we thought that it was necessary to use relative echogenicity calculated as a ratio to another organ when measuring and quantifying echogenicity. The spleen is rarely the site of primary diseases [30], and its echo structure is homogeneous [31]. Yabuki et al. reported that, in cats, the kidney/spleen ratio is a more constant method for quantitative evaluation of renal echogenicity than the kidney/liver ratio [32]. Thus, given these points, relative renal echogenicity as a ratio in reference to the spleen in the same field of view was used in the present study. The details of mechanism of increase of echogenicity in AKI remain unknown. The renal pathological change in AKI is known that epithelial cell detachment, cell swelling, epithelial casts, and loss of brush border in the light microscopy’s observation. In our study, the elevation of these histological changes after IRI was around 1–3 days after IRI [11], and was inconsistent with the peak of renal echogenicity. Furthermore, there were significant histological

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Fig. 6  Ultrasound images obtained 28 days after 45  min (U45) (a) and 60 min (U60) (b) of unilateral ischemia. Graph shows (c) echogenicity at multiple timepoints after IRI. Echogenicity of U45 (filled square) decreases to levels near that of Sham (empty circle), while

U60 (empty square) maintains high echogenicity (*P < 0.05 versus Sham at the same timepoint, †P < 0.05 versus U45 at the same timepoint). The vertical bars of the graphs are error bars showing standard deviation

changes in the kidney tissue 24 h after 30 min of bilateral ischemia, even though renal echogenicity was decreased to levels similar to that of Sham. These results indicated that there might be more microscopic histological damage that could not be observed with an optical microscope. For the AKI, damage is known that mitochondria swelling and cytoplasmic extrusion into the lumen in the electron microscopy observation, the increase of renal echogenicity might be referred to the electoron microscopic damage. Alternatively, increase of renal echogenicity may be associated to blood flow. The correlation between renal damage and reperfusion blood flow in IRI has been reported [15, 33]. There might also be dynamic factors such as blood flow which have some contribution to increase renal echogenicity in IRI. Increase of echogenicity is a complex phenomenon involving multiple causative factors. Further research is required for the elucidation for the mechanism of increase of echogenicity in AKI. There are some problems when applying quantified renal echogenicity to clinical practice. First, in humans, there is no good baseline echogenicity study, because the ischemic insult has already occurred by the time the patient presents for imaging. Second, it is difficult to compare the echogenicity of the entire kidney with that of the spleen in some patients, as they both cannot be placed in the same field of view due to organ location and body habitus. In such patients, there is no choice but to take ultrasonographic images of the kidney and spleen in different fields of view.

We think that accurate measurement over time may still be possible even in such patients if we pay attention to not changing the probe and the settings of the ultrasound device (such as gain, frequency, dynamic range, focus, mode, etc), and we have to put the probe at the same position of the body surface with marking and as much as possible take the same image using an organ landmark such as the hilum. Third, there are a number of underlying contributors to AKI [34, 35]. Our future research will examine the echogenicity in other AKI conditions, such as secondary to sepsis that is most common cause of AKI. Since there are some problems such as the above, further study is needed to address them.

Conclusions This study demonstrated the applicability of renal echogenicity as a noninvasive biomarker in a mice model of ischemia-induced AKI. Thus, quantified renal echogenicity may have potential for the evaluation of AKI and CKD. Acknowledgements  The scientific guarantor of this publication is Noriyuki Sugiyama, PhD. The authors of this manuscript declare no relationships with any companies, whose products or services may be related to the subject matter of the article. This work was supported by a Grant-in-Aid for Scientific Research (C, 26461245) from Ministry of Education, Culture, Sports, Science and Technology of Japan. No complex statistical methods were necessary for this paper.

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Institutional Review Board approval was not required, because this study was only on animal. Approval from the institutional animal care committee was obtained. We are grateful to Dr. Atsushi Yoden, board certified fellow of the Japan Society of Ultrasonics in Medicine (Osaka medical College, Department of Pediatrics), for the valuable suggestions and excellent technical advice regarding ultrasound. Compliance with ethical standards  Conflict of interest  The authors declare that they have no competing interest.

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