Int Urol Nephrol DOI 10.1007/s11255-015-0998-9

UROLOGY - ORIGINAL PAPER

Current radiological techniques used to evaluate unilateral partial ureteral obstruction: an experimental rabbit study Mehmet Yazıcı1 · Suleyman Celebi1,2 · Özgür Kuzdan1 · Hüseyin Koçan3 · Halil Suat Ayyıldız4 · I˙lkay Koray Bayrak5 · Meltem Ceyhan Bilgici5 · Oktay Yapıcı6 · Mehmet Kefeli7 · Ender Arıtürk8 

Received: 18 March 2015 / Accepted: 20 April 2015 © Springer Science+Business Media Dordrecht 2015

Abstract  Aim  The aim of this study was to evaluate functional and prognostic benefits of Doppler ultrasonography (DU), diuretic renal scintigraphy (DRS), and magnetic resonance urography (MRU) during diagnosis and follow-up of ureteropelvic junction obstruction (UPJO) and to examine apoptosis rates caused by UPJO in an experimental rabbit model. Method  Twenty-four rabbits were divided randomly into two groups. The left kidneys of 15 rabbits from the first

* Suleyman Celebi [email protected] Mehmet Yazıcı [email protected] Özgür Kuzdan [email protected] 1

Department of Pediatric Surgery, Kanuni Sultan Süleyman Education and Research Hospital, 34300 Istanbul, Turkey

2

Tahtakale mah. T32 cad. Bizimevler 4 C2, D:48 Ispartakule‑ Avcılar, Istanbul, Turkey

3

Department of Urology, Kanuni Sultan Süleyman Education and Research Hospital, 34300 Istanbul, Turkey

4

Department of Pediatric Surgery, Medipol University Faculty of Medicine, Istanbul, Turkey

5

Department of Radiology, 19 Mayıs University Faculty of Medicine, Samsun, Turkey

6

Department of Nuclear Medicine, 19 Mayıs University Faculty of Medicine, Samsun, Turkey

7

Department of Pathology, 19 Mayıs University Faculty of Medicine, Samsun, Turkey



8



Department of Pediatric Urology, 19 Mayıs University Faculty of Medicine, Samsun, Turkey

group underwent Ulm–Miller surgery to create UPJO, whereas the left kidneys of nine rabbits from the second group underwent sham surgery. A pressure flow study (Whitaker’s test) was done during postoperative week 6. Based on the Whitaker test, the DU, DRS, and MRU findings were compared. The number of apoptotic renal cells was counted after death. Result  The Whitaker test run during postoperative week 6 revealed obstructions in 15 rabbits from group 1; the nine rabbits of the sham group had no obstructions. Sensitivity and specificity of DRS were 93.3 and 88.8 %, respectively, and those of MRU were 93.3 and 88.8 %, respectively. The postoperative mean RI values were significantly higher than the preoperative values, associated with sensitivity of 86.6 % and specificity of 77.5 % for detecting UPJO. DRS, MRU, and RI could not predict UPJO in one (8 %), one (8 %), and two (16 %) kidneys, respectively. Likelihood ratio (LR) was 8.4 for MRU and scintigraphy, while for RI, LR was 3.9. Pathology specimens revealed that all kidneys with UPJO underwent apoptosis, and the number of apoptotic cells was significantly higher on the UPJOcreated side than on the contralateral and in the sham group (p 25 cm H2O was considered obstructive,  0.7) in the affected kidney, which is considered an obstruction [11]. Rabbits were placed on a scanning table in the supine position for scintigraphy. Hydration was initiated (10 ml/ kg body weight) 30 min prior to the study. 99mTc-mercaptoacetylglycylglycylglycine (MAG3) was injected intravenously at a dose of 1.85 MBq/kg, and a standard renal scan was performed [12]. After 20 min, 1 mg/kg furosemide

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was injected intravenously. The T½ was defined as the time when a 50 % decrease in kidney activity was observed after injecting furosemide. Scintigrams were recorded in the supine position using a gamma camera (Genesys, ADAC Laboratories, Milpitas, CA, USA). The renogram curve pattern, the ½ Tmax value, and the split function for both kidneys were considered in the evaluation. An obstructive hydronephrosis was assumed when T½ > 20 min [12]. One experienced technologist calculated the renal function parameters and generated renograms. The rabbits were placed on a scanning table in the supine position for MRU (13). An intravenous (IV) catheter was set before the study. Hydration was initiated (10 ml/ kg body weight) 30 min prior to the study, and 8 ml/kg/h hydration was maintained throughout the study. MRU was performed with a 1.5-T Siemens Symphony (Siemens Medical Solutions, Erlangen, Germany) using a phased-array body coil. Axial and coronal two-dimensional truffle (TR = 4.3 ms, TE = 2.1 ms) sequences were used for evaluation before the contrast agent was given. The change over time in the mean intensity within the selected area of all renal parenchymal renogram curves was determined to make a functional assessment. Urinary excretion of all kidney was evaluated from the renogram curve, and renal transit time (RTZ) was calculated. Accordingly, an obstruction of the kidney was considered to have occurred if the accumulation consisted of all kidney renogram curve in the renal pelvis was minimally visually washed or delayed. The MRU images were evaluated by two radiologists, and the results were determined by consensus. A RTZ of 8 min was accepted as an obstruction [13]. The RI and MRU were evaluated by an experienced radiologist, and DRS was evaluated by a nuclear medicine physician. The rabbits were killed after radiology, both kidneys were excised, a vertical cut from the dorsal part of the corticomedullary region to the renal pelvis was made, and renal apoptotic cells were counted using the terminal deoxynucleotidyl transferase dUTP nick end-labeling method [14]. In addition, sensitivity and specificity of scintigraphy, RI, and DMR were evaluated based on the pressure flow

study results. The associations among pressure flow, scintigraphy, MRU, and RI were assessed using likelihood ratio analysis. The extent of apoptosis and the numbers of apoptotic cells in the left and right kidneys were compared using Wilcoxon’s signed-rank test. The Mann–Whitney U test was used to compare the numbers of apoptotic cells in the kidneys of unobstructed and obstructed rabbits.

Results The pressure flow study revealed UPJO in 15 rabbits, and all nine rabbits in the sham group were unobstructed. The sensitivity of scintigraphy compared to pressure flow was 93.3 % and specificity was 88.8 %, whereas MRU had sensitivity and specificity of 93.3 and 88.8 %, respectively, compared with pressure flow (Table 1). The mean renal RI in rabbits on the UPJO side was significantly higher postoperatively than preoperatively. The RI sensitivity and specificity were 86.6 and 77.5 %, respectively, compared with those of the pressure flow (Table 1). Urinary tract dilatation was observed in all rabbits on USG, and the results mostly correlated with scintigraphy and MRU. MRU was successful in identifying the pathology in all UPJO-created rabbits, except one. Accordingly, the sensitivity in determining the etiology of dilated compared with non-obstructive (sham) operative findings on MRU was calculated as 93.3 %. Scintigraphy failed to find an obstruction in one rabbit. MRU and DRS were similarly effective for the functional evaluation. The RI was normal in two kidneys of two rabbits with UPJO, so the obstruction was misidentified. A likelihood ratio (LR) greater than 1 indicates that the test result is associated with the disease; the greater the value, the more convincingly the finding suggests that disease. Three diagnostic techniques had values greater than 1. LR = 8.4 for MRU and scintigraphy, which means that these are strong diagnostic criteria, while for RI, LR = 3.9, which means that this is a moderate diagnostic criterion. All UPJO-created rabbits had kidney cells undergoing apoptosis. The UPJO side showed an increased number of apoptotic cells, mainly localized to focal lesion areas

Table 1  Diagnostic value of the tests used for detecting ureteropelvic junction obstruction and the rates of apoptosis Whitaker (n) Mag3 scintigraphy (n) MR urography (n) Resistivity index (n) Apoptosis (n) 15

14

14

13

15

9

8

8

7

0

PPV NPV

93.3 % 88.8 %

93.3 % 88.8 %

86.7 % 77.8 %

LR (+)

8.4

8.4

3.9

Detection rate of UPJO group (n) Detection rate of non-UPJO group (Sham) (n)

Likelihood ratio

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A

B2

B1

B1

C1

C2

B2

C2

D2

D1

Fig. 2  Diagnostic studies in experimental rabbit ureteropelvic junction obstruction model. a Pressure flow study (Whitaker test) to identify ureteropelvic junction obstruction (UPJO). b Diuretic renal scintigraphy and 99mTc-MAG3 show 1 normal renal scintigraphy in the sham group and 2 UPJO in the left kidney. c Magnetic resonance

urography and T2-weighted sequence of the 1 sham group and 2 kidney with UPJO (red line, left kidney with UPJO; black line, right kidney). d Kidney histology after killing the rabbits showing 1 normal tubular cells (×400) and 2 TUNEL staining for tubule epithelial apoptotic cells (arrow) at the UPJ (×400)

Table 2  Apoptosis rates in rabbits with a ureteropelvic junction obstruction

Discussion

Apoptosis UPJO-created kidney (left side) Contralateral side of UPJO-created kidney (right side) Explored kidney of sham group (left side) Opposite side of explored kidney at sham group (right side)

2.75 ± 1.09 0.89 ± 0.72 0.42 ± 0.59 0.43 ± 0.67

P apoptosis obst—sham

p = 0.0001

P apoptosis obst—contralateral

p = 0.001

(Fig. 2). The apoptosis rate was significantly higher in the UPJO-created group than that in the sham group. The total apoptotic cell count in obstructed rabbits (left kidney) was significantly higher than that in the contralateral unobstructed side (right kidney) (Table 2). DRS, MRU, and RI could not predict UPJO that concluded with apoptosis in one (8 %), one (8 %), and two (16 %) kidneys, respectively. Despite the increased level of apoptosis on the opposite side of UPJO-created kidneys, no significant difference was observed compared with the sham group (p = 0.09).

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UPJO is a blockage or obstruction of urine flow from the kidney to the proximal upper ureter and is the most common cause of postnatal hydronephrosis [6]. UPJO is a functional obstructive condition, caused by defective fetal smooth muscle/nerve development at the kidney level, with lack of peristaltic wave propagation caused by the presence of an aperistaltic segment [4]. In UPJO, urine ejection from the renal pelvis to the ureter is limited, which leads to increased kidney back pressure, hydronephrosis, and progressive deterioration of kidney function. Therefore, it is important to understand how to diagnose and treat the condition [6]. We used the Ulm–Miller technique to create experimental UPJO. The Ulm–Miller method was first used in 1962 by Ulm Aaron Hardy and Frank Miller to create hydronephrosis [9]. A UO is created by burying the ureter in the psoas muscle, which causes rapid expansion of the pelvis, but renal function does not deteriorate despite prolonged obstruction of the pelvis. It is believed that this method causes mild-to-moderate hydronephrosis dilatation, as seen in cases of antenatal and neonatal hydronephrosis [9]. We

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demonstrate that all rabbits that underwent the Ulm–Miller surgery developed clinical UPJO. If UPJO is not diagnosed in childhood, the chronic obstructive process may induce more apoptosis and reduce renal function [15]; thus, there is increasing interest in the use of different diagnostic modalities, including urinary markers and USG with various related tests, including calculations of RI, DRS, and MRU. However, the sensitivities of these techniques are not always acceptable for diagnosing renal injury related to UPJO [16]. In our results, at least one kidney (8 %) did not exhibit radiological findings of UPJO, despite the presence of significant apoptosis. Many methods can be used to separate obstructive dilatation from unobstructive dilatation, including a pressure flow study (Whitaker test) [10]. The test directly measures ureteral resistance by recording the pressure gradient across the suspected area of obstruction. Whitaker worked with 170 obstructed/unobstructed cases and experienced difficulty differentiating the two types of cases using conventional diagnostic methods; thus, he developed the test that bears his name [10]. Performing the Whitaker test requires placing a catheter in the bladder as well as an antegrade pyelogram needle in the kidney. Contrast is delivered at a constant rate through the needle into the kidney, simulating diuresis, and pressures in the kidney and the bladder are measured. Although sensitivity of the test is nearly perfect [17], it is not recommended for children because it is invasive and requires anesthesia to insert the pressure lines [17]. We used the Whitaker test and compared the sensitivities and specificities of other conventional techniques to those of the Whitaker test. USG is the imaging modality of choice for diagnosing hydronephrosis but cannot be used to differentiate between obstructed and unobstructed kidneys [18]. Doppler USG and calculating the related parameters such as RI provide additional evidence for diagnosing complete or acute obstructive uropathy [19]. The RI is the peak systolic velocity minus the lowest diastolic velocity divided by the peak systolic velocity. Patients with an RI > 0.7 exhibit an obstructive pattern on DRS [19]. However, we found that sensitivity and specificity of this conventional technique were insufficient to predict the presence of an obstruction leading to apoptosis. MAG3 scanning is essential to distinguish an obstructed renal pelvis from a renal pelvis that is dilated but otherwise normal [12]. MAG3 scanning reveals differences in renal function by comparing isotope uptake between the two kidneys, which reflects renal blood flow. The decisive investigation is renal function scintigraphy, which not only shows renal function on both sides but is also especially important for determining the flow relationships in the urinary tract collection system. Some indications for surgery include 20-mm anterior–posterior diameter of the renal pelvis on USG, pain, and infection [20]. Although MAG3 scanning is a basic test in most clinics, we found that MAG3 scanning afforded a high but incomplete sensitivity when it was used to determine whether a kidney was undergoing apoptosis. MRU evaluates renal blood flow, anatomy, and urinary excretion [13]. MRU provides an anatomical image superior to those of USG or DRS. Rohrschneider et al. [13] used DRS and MRU to study 20 pigs and found a close correlation between the results of the two techniques. Perez-Brayfield et al. studied 71 children and used the MRU results to describe renal function as determined by DRS. They found a high correlation between the results of the two techniques [8]. In our study, sensitivity and specificity of DRS and MRU were similar; however, both yielded some false-negative results. DRS and MRU could not predict UPJO that concluded with apoptosis in one (8 %) and one (8 %), respectively. Misdiagnosis was at different rabbits. This finding convinces us that if both tests are conducted together, a complete diagnosis can be established by estimating the apoptosis rate caused by UPJO. Obstruction of the upper tract during the acute phase leads to increases in ureteric and renal pelvic pressures. A progressive rise in renal pelvic pressure from 20 to 40 cm H2O in pediatric patients causes a hydrodynamic imbalance followed by transmural compression of peritubular afferent arterioles with subsequent renal hypoperfusion [21]. If the obstructive uropathy continues, the interaction between the cells and cell matrix at the distal tubules and collecting ducts results in an increase in the numbers of apoptotic tubular and interstitial cells [22]. Thus, renal tubular proliferation is suppressed and apoptosis increases [22]. Apoptosis of the tubular cells seems to be the main cause of the tubular atrophy together with epithelial–mesenchymal phenotypic transdifferentiation [23]. All of these changes damage the renal parenchyma, leading to sustained loss of renal function [23]. Chevalier et al. [24] revealed that the nephron loss and residual injury persisting following the period of early postnatal obstruction lead to progressive renal damage. Special focus is given to the role of apoptosis as a major cause of cell death during and after a complete ureteric obstruction. Tubular cell apoptosis is an early event that occurs before the onset of frank fibrosis; thus, reducing the initial cell death may prevent progression to fibrosis [25]. The numbers of tubular apoptotic cells increased over 15 days and those of apoptotic interstitial cells rise over 6 weeks [26]. In this study, we demonstrate that apoptosis was mainly localized to the tubular epithelial and interstitial cells in cases of unilateral ureteral obstruction. Apoptosis was an inevitable result 6 weeks after formation of UPJO.

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Despite widespread use of prenatal USG, a certain number of symptomatic UPJO are still detected later in infancy. The surgical decision is relatively easier in these patients, and functional outcomes seem to be more satisfactory [26]. As in our model, we did not choose neonatal rabbits, as we thought we could obtain more accurate findings if the kidneys completed maturation. Follow-up using a single radiological technique can give rise to false-negative results. We believe that using different radiological techniques is more reliable for determining the prognosis during the follow-up of UPJ obstruction. Although we have made recommendations for appropriate screening based on our experimental study, these must be supported by multicenter prospective clinical studies. In conclusion; the aim of UPJO treatment is to prevent deterioration in renal function. However, the diagnosis of obstruction is equivocal in some circumstances, and no cutoff measure is available to confirm the presence or absence of an obstruction. Our results indicate that the extent of apoptosis may be underestimated if these techniques are used separately. New radiological techniques are required, associated with better methods for diagnosing an obstruction, or a combination of radiological techniques should be used to aid in the making of management decisions. Conflict of interest  Authors declare that there is no conflict of interest.

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Int Urol Nephrol 9. Ulm AH, Miller F (1962) An operation to produce experimental reversible hydronephrosis in dogs. J Urol 88:337–341 10. Whitaker RH (1979) An evaluation of, 170 pressure flow studies of the upper urinary tract. J Urol 121:602–604 11. Riccabona M, Ring E, Fueger G, Petritsch P, Villits P (1993) Doppler sonography in congenital ureteropelvic junction obstruction and multicystic dysplastic kidneys. Pediatr Radiol 23:502–505 12. Mandell G, Cooper J, Leonard J (1997) Procedure guideline for diuretic renography in children. J Nucl Med 38:1647–1650 13. Rohrschneider WK, Becker K, Hoffend J, Clorius JH, Darge K, Kooijman H, Tröger J (2000) Combined static-dynamic MR urography for the simultaneous evaluation of morphology and function in urinary tract obstruction. II. Findings in experimentally induced ureteric stenosis. Pediatr Radiol 30(8):523–532 14. Negoescu A, Guillermet C, Lorimier P, Brambilla E, Labat Moleur F (1998) Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity. Biomed Pharmacother 52(6):252–258 15. Choi YJ, Baranowska-Daca E, Nguyen V, Koji T, Ballantyne CM, Sheikh-Hamad D et al (2000) Mechanism of chronic obstructive uropathy: increased expression of apoptosis-promoting molecules. Kidney Int 58(4):1481–1491 16. Mitterberger M, Pinggera GM, Neururer R et al (2008) Comparison of contrast-enhanced color Doppler imaging (CDI), computed tomography (CT), and magnetic resonance imaging (MRI) for the detection of crossing vessels in patients with ureteropelvic junction obstruction (UPJO). Eur Urol 53:1254–1262 17. Wahlin N, Magnusson A, Persson AE, Lackgren G, Stenberg A (2001) Pressure flow measurement of hydronephrosis in children: a new approach to definition and quantification of obstruction. J Urol 166:1842–1847 18. Heinlen JE, Manatt CS, Bright BC, Kropp BP, Campbell JB, Frimberger D (2009) Operative versus nonoperative management of ureteropelvic junction obstruction in children. Urology 73:521–525 19. Rawashdeh YF, Mortensen A, Horlyck J et al (2003) Resistive index: an experimental study of acute complete unilateral ureteral obstruction. Invest Radiol 38:153–158 20. Ransley PG, Dhillon HK, Gordon I, Duffy PG, Dhillon MJ, Barrat TM (1990) Postnatal management of hydronephrosis diagnosed by prenatal ultrasound. J Urol 144:584–587 21. Wen JG, Chen Y, Frøkiaer J et al (1998) Experimental partial unilateral ureter obstruction. I. Pressure flow relationship in a rat model with mild and severe acute ureter obstruction. J Urol 160(4):1567–1571 22. Stratta P, Canavese C, Dogliani M, Mazzucco G, Monga G, Vercellone A (1991) The role of free radicals in the progression of renal disease. Am J Kidney Dis 17:33–37 23. Chevalier RL, Thornhıll BA, Forbes MS et al (2010) Mechanisms of renal injury and progression of renal disease in congenital obstructive nephropathy. Pediatr Nephrol 25:687–697 24. Chevalier R, Thornhill B (2000) Chang a unilateral ureteral obstruction in neonatal rats leads to renal insufficiency in adulthood. Kidney Int 58:1987–1995 25. Kennedy WA 2nd, Stenberg A, Lackgren G et al (1994) Renal tubular apoptosis after partial ureteral obstruction. J Urol 152:658–664 26. Cain MP, Rink RC, Thomas AC, Austin PF, Kaefer M, Casale AJ (2001) Symptomatic ureteropelvic junction obstruction in children in the era of prenatal sonography: is there a higher incidence of crossing vessels? Urology 57:338–341

Current radiological techniques used to evaluate unilateral partial ureteral obstruction: an experimental rabbit study.

The aim of this study was to evaluate functional and prognostic benefits of Doppler ultrasonography (DU), diuretic renal scintigraphy (DRS), and magne...
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