Abstract
Background/Aim: This study performed 99mTc-MAG3 renal scintigraphy on rabbit kidneys and evaluated its ability to identify obstructive or non-obstructive kidneys. Materials and Methods: Renal function was assessed during a four-week post-obstruction period by obtaining planar images of 99mTc-MAG3 activity following an ear vein injection. The individual renal function was evaluated by renal scintigraphy in conjunction with histopathological and morphological examinations. Results: The renal perfusion of 99mTc-MAG3 in the right kidney with a ureteral obstruction decreased with time. The width, height, and cortical thickness of the obstructed right kidney were significantly larger than those of the left kidney. A histopathological examination four weeks after the ureteral obstruction revealed a typical pattern of urinary tract obstruction, including multiple tubules, enlargement of the interstitial area, and cytoplasmic vacuoles. Conclusion: 99mTc-MAG3 renal scintigraphy provides the kidney shape and size and can identify potential obstructive and non-obstructive kidneys in rabbits.
Urinary tract obstruction (UTO) is a condition of urination that can lead to progressive renal dysfunction. It is commonly associated with dysuria, is a frequent cause of kidney damage (1), and can result from a wide range of congenital, intraluminal, parietal, and extrinsic disorders that affect the urinary tract. Renal function should be assessed in both the early stages and during the follow-up of UTO (2).
Hydronephrosis (HN) is a condition in which one or both kidneys grow and swell when urine collects inside the kidneys and can result from several diseases. The conditions often associated with unilateral HN include acute/chronic unilateral obstructive uropathy, ureter obstruction due to scars, tumors and uroliths, vesicoureteric reflux illness, and kidney stones.
Biochemical and histopathological assays are used to diagnose and characterize renal damage. Blood and urine tests can measure the overall kidney function but not the individual kidney function. The best indicator of the kidney function is believed to be the glomerular filtration rate (GFR). However, the direct measurement of GFR is not feasible because the filtration process occurs simultaneously in millions of glomeruli (3). Renal function is revealed by diagnostic imaging methods, such as magnetic resonance imaging, computed tomography (CT), positron emission tomography (PET), venous pyelography, and renal or abdominal ultrasonography (3). Despite this, these methods cannot evaluate the functional level of individual kidneys. In patients with suspected renal disease, intravenous contrast urography may provide more information on the functionality, but this is a laborious procedure, and the x-ray contrast may be nephrotoxic.
Under normal conditions, animals have sufficient renal function to survive with only one functioning kidney. Unfortunately, this is not always the case for animals with kidney damage. In some cases, the ability to measure the individual renal function is important because such cases may include the prospect of nephrotomy or nephrectomy (4). An assessment of the individual renal function can have a significant impact on the plan to surgically remove kidney stones or the entire kidney damaged by an infection or tumor (5). Non-invasive means of determining renal function in individual kidneys may be advantageous during planning for medical management or surgical intervention of renal diseases.
Renal nuclear medicine imaging techniques offer a combination of information on the morphological aspects of the kidneys, and their functionality (4). 99mTc-labelled radiopharmaceuticals are highly suitable for kidney disorders, because of their superior imaging qualities, reasonable price, and excellent availability (6). MAG3 can be labeled with 99mTc, making it a very suitable tracer for imaging procedures.
Nephrography is often performed to determine the differential renal function of patients undergoing or being evaluated for urologic surgery (2). The principle of renal scintigraphy is based on the intravenous administration of radiopharmaceuticals accumulated and excreted by the kidneys. Radiopharmaceuticals can be detected externally with a gamma camera (1). The renogram monitors the arrival and release of radiopharmaceuticals in a dynamic sequence, allowing an estimation of the renal function. Non-invasive means for determining the individual renal function may be advantageous for the medical management or surgical intervention of renal disease (3). Quantitative renal scintigraphy can assess the renal function that may persist after such surgical procedures (7).
Functional and morphologic studies with renal scintigraphic scans play important roles in diagnosing and following up various renal disorders (8). 99mTc-DMSA renography is considered the standard method for assessing the renal cortex for over 40 years (9). Some physicians have reported 99mTc-MAG3 as an alternative to 99mTc-DMSA in most renal disorders in pediatric patients (1). Up to 90% of 99mTc-MAG3 is excreted mainly by tubular secretion, and the rest is excreted from the blood by glomerular filtration. A high percentage of the injected 99mTc-MAG3 is bound to the plasma proteins, and is mainly reversible. This study examined the normal or abnormal renal function by performing 99mTc-MAG3 scintigraphy using a rabbit model. Here, 99mTc-MAG3 was used as a radiopharmaceutical to image the cortical kidney. In addition, histopathological and morphological examinations of the rabbit kidney were performed in parallel to support the renal scintigraphy findings.
Materials and Methods
Experimental animals. Twelve healthy male New Zealand white rabbits (age: 12 weeks, body weight: 3.1-3.2 kg) were divided randomly into two groups of six animals each: saline-injected control and 99mTc-MAG3-injected groups. Each rabbit was maintained in a separate cage in an air-conditioned room (temperature 20±2°C, humidity 40~60%) with illumination between 08:00 and 20:00 at the Laboratory Animal Research Center (Cheongju, Republic of Korea). The rabbits were anesthetized by intramuscular administration of a cocktail of 35 mg/kg ketamine HCl (Yuhan Pharma Co., Seoul, Republic of Korea) and 5 mg/kg xylazine (Rompun®, Bayer Korea Co., Republic of Korea). For ureter obstruction, each rabbit underwent general anesthesia before the proximal part of the right ureter was ligated with 2-0 silk at two places adjacent to the renal pelvis. All animals received antibiotics intramuscularly (20 mg/kg amoxicillin, Foxoline injection, SamJin Pharma Co., Seoul, Republic of Korea) and analgesics (3 mg/kg tramadol, Tamador injection, DongKwang Pharma Co., Seoul, Republic of Korea) for seven days, respectively. The Animal Care Committee of Chungbuk National University, Republic of Korea, approved all protocols employed in this study (CBNU A-19-23).
99mTc-MAG3 renal scintigraphy. Renal scintigraphy was performed before surgical intervention, immediately after surgery, and at one, two, three, and four weeks post-ureteral ligation. The rabbits were administrated atropine (0.05 mg/kg) subcutaneously. Anesthesia was induced with xylazine (1.1 mg/kg) and maintained with ketamine (11 mg/kg). Data were obtained using a dual-head gamma camera with a CT capacity (Infinia Hawkeye, GE Healthcare, Chicago, IL, USA). Scintigraphy was performed after an ear vein injection of 3 mCi 99mTc-MAG3 (TechneScan MAG3, Mallinckrodt Diagnostica, the Netherlands) or saline physiological solution. The scintigraphic images were recorded every 2 s for 1 min and then every 1 min for 25 min. The percentage of kidney function was calculated from anterior and posterior images of a 99mTc-MAG3 scan and by drawing regions of interest around each kidney and around the background area of each kidney.
Histopathological and morphological assays. All rabbits were euthanized by an intramuscular administration of butorphanol tartrate (0.1 mg/kg, MyungMoon Pharm Co., Seoul, Republic of Korea) and acepromazine maleate (0.8 mg/kg, SF Co., AnSan, Republic of Korea). After each kidney was cut from the rabbits, the width, height, and thickness were measured with calipers. At four weeks after ureter ligation, all rabbits were euthanized and the kidneys were removed followed by overnight fixation in 10% buffered formalin. Subsequently, paraffin tissue sections were stained with hematoxylin and eosin using standard procedures.
Statistical analysis. Statistical analysis was performed using SPSS software (version 19.0.1.1; IBM SPSS Statistics for Windows; IBM Corp., Armonk, NY, USA). Data are presented as the mean ± standard deviation values. The normality and homogeneity of the data were confirmed before performing an analysis of variance (ANOVA). The differences among experimental groups were assessed by one-way ANOVA followed by Duncan’s multiple range tests. The null hypotheses of no significant difference were rejected if the obtained p-values were less than 0.05.
Results
Renal uptakes of 99mTc-MAG3. Time-radioactivity curve (TRC) analysis revealed radiopharmaceutical uptake in both kidneys after a bolus injection of 99mTc-MAG3. The renal uptakes of 99mTc-MAG3 decreased rapidly in the right kidney with ureteral obstruction but increased relatively in the contralateral left kidney. An abnormal curve was observed in the uptake of 99mTc-MAG3 in the occluded right kidney at a level that decreased gradually from one minute after injection (Figure 1). Renal perfusion of 99mTc-MAG3 was reduced significantly in the obstructive kidney, which is indicated by the hypoactivity, and further decreased as the disease progressed (Figure 1). Therefore, the renal uptake of 99mTc-MAG3 in the obstructive kidney decreased with time, indicating that it was not excreted from the kidney.
99mTc-MAG3 renography of a rabbit model with right ureteral obstruction. (A) Regions of interest are drawn around the right and left kidneys. (B) Time-radioactivity curves for left (top, red line) and right kidneys (bottom, green line).
Morphological and histopathological assays. The morphology of the kidneys was compared. The ureter-obstructed right kidney was larger than the normal left kidney. The left kidney maintained a consistent size during the experimental periods, but the obstructed right kidney became progressively larger (Figure 2A). A histopathological examination four weeks after the ureteral obstruction showed a typical pattern of unilateral UO, including multiple tubules, enlargement of the interstitial area, and cytoplasmic vacuoles. The renal pelvic space was expanded in the obstructed kidneys, and the components of the renal medulla, particularly the inner medulla and papilla, decreased as the disease progressed. The tubulointerstitial region of the ureter-obstructed right kidney appeared markedly degenerated, but no apparent structural changes were observed in the contralateral kidney (Figure 2B). The right kidney at four weeks post-surgical intervention exhibited HN. Four weeks after ureteral obstruction, the size of the obstructed right kidney increased and, width, height, and cortical thickness were significantly larger than those of the left kidney (Figure 3) (p<0.05).
Images of the kidney in a rabbit model of right ureteral obstruction at 4 weeks. (A) Macrophoto shows hydronephrosis in the right kidney. Microphoto shows cell bodies in renal tubules (B, white arrow head, ×100) and glomerular condensation and pyknosis (C, black arrow, ×200). H&E stain.
Comparison of width, height, and cortical thickness of kidneys in rabbits four weeks post-ureter obstruction. n=6, *p<0.05.
Discussion
The ureter is a bilateral fibromuscular duct that carries urine from the kidney to the urinary bladder. The movement of urine is due to peristalsis, which is transmitted from one muscle cell to the next. Ureteral obstruction (UO) is a condition of dysuria that can cause progressive renal dysfunction and is a frequent cause of renal damage (1). In particular, UO is a significant cause of renal dysfunction in neonatal and infants (10, 11).
Radionuclide research is important in diagnosing and following up various kidney diseases (4). Non-invasive methods for diagnosing renal function may be advantageous for surgical intervention or medical treatment when suffering from renal diseases. A previous study reported renal scintigraphy using various radionuclides such as 99mTc-MAG3, 99mTc-labeled diethylenetriaminepentaacetate (99mTc-DTPA), and 99mTc-labeled dimercaptosuccinic acid (99mTc-DMSA) (1, 4). 99mTc-labeled radiopharmaceuticals are suitable for kidney disorders because of their superior imaging qualities, reasonable price, and excellent availability (6). The imaging provided by each radionuclide reflects the various renal functional parameters (12). Although these radionuclides accurately measure all renal functional parameters, there are some differences due to the different biological properties of radionuclides (13). Radionuclides have different plasma protein and plasma clearance levels, depending on the mechanism of renal excretion combined with the retention of radioactive material in renal cells (14).
In diagnostic imaging, 99mTc-MAG3 provides high-quality renal images and is widely recognized as an excellent renal radiopharmaceutical. In patients with markedly impaired renal function, 99mTc-MAG3 can be used instead of 99mTc-DTPA to assess renal function due to the faster clearance and higher extraction rate (15). Because 99mTc-MAG3 is excreted by renal tubular excretion, the renal image is more prominent than a 99mTc-DTPA renal examination, which is useful for a follow-up examination before the diagnosis and treatment of patients with UTO (1). The important feature of 99mTc-MAG3 is that it can be cleared from the blood by the renal tubules and excreted to the collecting system (16). This allows an assessment of renal perfusion, tubular function, tubular secretion to the collecting system, and subsequent drainage. Images recorded after an injection of 99mTc-MAG3 reflect the tracer concentration in the renal tubules, and high-resolution images provide an accurate assessment of the renal cortex integrity (16).
CT, ultrasound, and x-rays show the size and shape of the kidney, but not its function. 99mTc-MAG3 scintigraphy can also observe renal blockages, obstructions, or damage (11). 99mTc-MAG3 remains in the intravascular space and has a high target-to-background ratio and good image quality, particularly when renal function is low or immature (1). Renal scintigraphy with 99mTc-MAG3 has been used widely to assess post-operative complications in renal transplant recipients (17). Furthermore, this is preferred for neonate patients with renal dysfunction because of the more efficient extraction. In the present study, 99mTc-MAG3 renal scintigraphy evaluated renal size and shape by the renal uptake. In addition, renal uptakes of the 99mTc-MAG3 were compared in rabbits with ureteral obstructive or non-obstructive kidneys.
Histopathological findings are used to diagnose and characterize renal disorders. A morphological assessment of the kidney is an objective method for determining the structure and function of the kidney. The histopathological examination showed that the ureter-obstructed kidney had a minimal degree of interstitial inflammation with mild dilation of the collecting tubules, flattening, and atrophy (18). These results suggest that the renal functioning of the rabbits with an obstructed ureter was impaired. This study is consistent with previous reports (4).
Although blood and urine exams cannot measure the individual renal function, scintigraphy provides important information regarding the visualization of the collecting system of the kidney. Renal scintigraphy avoids unnecessary additional radiation to children and saves time. This study was consistent with the report that 99mTc-MAG3 may evaluate the renal cortex quickly (18).
In renal scintigraphy, the TRC showed radiopharmaceutical radioactivity uptake after 99mTc-MAG3 administration. The TRC was determined in each kidney, and renal perfusion was assessed by the renal uptakes of 99mTc-MAG3. The renal uptakes of 99mTc-MAG3 increased gradually in the non-obstructive kidney but decreased gradually in the obstructive kidney. Therefore, 99mTc-MAG3 renography can provide a differential diagnosis between obstructive and non-obstructive kidneys. Given these characteristics, renal scintigraphy is a useful tool for evaluating post-operative complications in renal transplant patients (19). The long residence time of 99mTc-DMSA in the renal cortex leads to high radiation doses to the neonatal and infant patients (18). This discomfort is one of the major limitations in diagnostic methods. Outpatients are often inconvenient because they need to visit the hospital twice to administer 99mTc-DMSA and the scan. The low energy and relatively short half-life of 99mTc-MAG3 are advantages for keeping the radiation burden low for the patient and practitioner. Therefore, 99mTc-MAG3 is good for evaluating renal function in neonatal and infants in a short time. The TRC of 99mTc-MAG3 enables a quantitative evaluation of multiple features of renal function, and the renography provides information on the kidney morphology. The TRC increases linearly as 99mTc-MAG3 flows into renal tissue, while the descending phase assesses the tubular function (20). The slope of the ascending phase after the renal uptake of 99mTc-MAG3 was increased in non-obstructive kidneys. Similar results were demonstrated in rat or dog models (11). The blood flow in post-arterial circulation was increased in the non-obstructive kidney.
This experiment investigated the renal uptake of 99mTc-MAG3 and compared the histopathological and morphological outcomes in each kidney in a rabbit model with unilateral ureter obstruction. Kidney function is traditionally investigated based on blood analysis or urinalysis. This study provides a minimally invasive, simple, and quantitative renal function test, which does not require blood or urine sampling.
These results suggest that a 99mTc-MAG3 renal scan can identify potential obstructive kidneys, a differential diagnosis between obstructive and non-obstructive uropathies, or HN in patients. Nevertheless, further investigation into GFR, effective renal plasma flow, or functional renal mass combined with functional and morphological data will be needed in the future.
Acknowledgements
This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01620801)” Rural Development Administration, Republic of Korea.
Footnotes
Authors’ Contributions
Lim conceived the original idea and processed the renograms. Choi provided the review and editing support. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received November 18, 2021.
- Revision received December 2, 2021.
- Accepted December 3, 2021.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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