Assessment of renal function in mice with unilateral ureteral obstruction using ^99mTc-MAG3 dynamic scintigraphy

Male C57BL/6N mice, aged 6 weeks, were purchased from Harlan Laboratories (Indianapolis, IN). Animals were fed a standard diet and allowed free access to water. All animal procedures were conducted in accordance with institutional guidelines and approved by the Vanderbilt University Institution of Animal Care and Use Committee.

Mice were anesthetized with 2% isoflurane and the left kidney was exposed through the site of the left flank incision. The ureter was obstructed completely near the renal pelvis using a 4–0 silk tie suture at two points. Sham-operated mice underwent the same surgical procedure except for the ureter ligation. The mice were imaged at 0 (3 hours), 1, 3, and 6 days after surgery.

Mice were anesthetized with 1.5%/98.5% isoflurane/oxygen mixture and two mice at a time were placed directly on top of a parallel-hole collimator positioned over one of the four camera heads of a NanoSPECT/CT (Bioscan, Washington, DC). The NanoSPECT was set up in single-camera planar-dynamic mode. A 40 min dynamic acquisition was initiated simultaneously with retro-orbital injection of ~37 MBq/0.2 mL of ^99mTc-MAG3 (obtained from VUMC Radiopharmacy, Vanderbilt University). The 40 min dynamic planar acquisition was divided into 240 frames yielding a temporal resolution of 10 sec. Two-dimensional images were collected in a 256 x 256 matrix with a pixel size of 0.29 mm x 0.29 mm. For static imaging, three dimensional images were collected in a 176 x 176 x 112 matrix with a pixel size of 0.2 mm x 0.2 mm x 0.2 mm through a 40-min scan (a temporal resolution of 40 min), producing high resolution imaging data. In a comparison study (retro-orbital vs. intravenous injections), ^99mTc-MAG3 was injected into mice via jugular catheter. Jugular vein catheterization was carried out as described [11].

The planar images were imported into the medical image viewer software Amide [12]. Two-dimensional ellipsoidal shaped regions of interest (ROIs) were drawn around each kidney and a background region outside the mouse. The radioactivity within the ROIs was extracted for each frame and normalized to the injected dose for each animal, dividing image voxel values by the actual injected dose as measured in a CRC-15W dose calibrator (Capintec, Ramsey, NJ), to establish the TACs over the entire duration of the scan. The data were expressed as normalized image value (NIV). The slope of initial linear uptake (from 10s to 40s)(SIU) in units of 1/s, peak activity in units of NIV, and the time to peak or plateau (TTP or TTPt) in units of seconds were computed from the renogram of each kidney [5, 13]. [Note: Since the TACs of UUO kidneys plateau and do not peak, we measured the plateau activity and the time-to-plateau for these kidneys.]

Structural changes in UUO kidneys were assessed by MRI studies on a Varian 7T horizontal bore imaging system (Lexington, MA). Anesthesia was induced and maintained with a 1.5%/98.5% isoflurane/oxygen mixture. A constant body temperature of 37°C was maintained using heated air flow. Animals were placed in a prone position and imaged using a birdcage 25-mm inner diameter radiofrequency (RF) coil. All imaging protocols were based on a field-of-view of 25.6 x 25.6 mm². A multi-slice gradient echo imaging sequence [repetition time (TR) =25ms; echo time (TE) = 2.5ms; 128 x 128 matrix; 2-mm slice thickness] was used to acquire 3-plane multi-slice images for proper positioning and slice selection. A coherent gradient echo sequence was used for T1-weighted imaging (TR = 45 ms; TE = 2.25 ms; flip angle = 35°; number of excitations (NEX) = 81). T2 contrast was achieved with the use of a fast spin-echo (FSE) sequence (TR = 2000ms; effective TE = 48 ms; RARE-factor 8, 256 x 256 matrix; 0.5-mm slice thickness).

First, we confirmed generation and severity of UUO model by MRI and histological investigation. Figure 1 shows representative T1- and T2-weighted MR images of a UUO mouse at 0, 1, 3 and 6 days post-surgery. The renal pelvic space was gradually expanded in obstructed kidneys as early as 1 day post ureteral ligation and the components of renal medulla, especially inner medulla and papilla, were remarkably decreased as the disease progressed. Reduction of renal cortex was also observed at day 3 and day 6 post obstruction. Histological investigation displayed typical patterns of UUO renal disease, including tubular dilatation, flattening and atrophy, and expansion of interstitial area with extracellular matrix accumulation and infiltration of inflammatory cells (Figure 1, bottom panels). Tubulointerstitial region progressed markedly over 6 days after ureteral obstruction. Obvious structural changes were not observed in contralateral kidneys and sham-operated mouse kidneys (data not shown).

Renal function was assessed in UUO kidneys based on the TACs and their parametric analysis. Three parameters were measured for individual kidneys: 1) the slope of initial linear uptake (SIU, 1/s), which is related to renal perfusion; 2) the peak activity (NIV); and 3) the time-to-peak or plateau (TTP or TTPt, sec) (Figure 2). Radiotracers are usually administrated via the tail vein in rodents. However, repeated injections are difficult as the procedure frequently causes soft tissue extravasations and tail scars, preventing subsequent injections. Repeatable and proper intravenous injections are fundamental for the longitudinal and quantitative analysis of ^99mTc-MAG3 renal scintigraphy. Therefore, we first compared retro-orbital and intravenous injections, given the facts that the former is much easier to accomplish at multiple time points and that retro-orbital injection was recently shown to be an effective route of radiotracer administration during PET studies in mice [14]. As shown in Figure 3A, the TACs of ^99mTc-MAG3 renal scintigraphy obtained from retro-orbital injections were comparable to those from intravenous injections via jugular vein catheter. There was no significant difference in the SIU, peak activity, and TTP parameters between these two procedures (Figure 3B). In addition, a comparative study using UUO day 1 mice also showed no difference between these two administration routes (data not shown). These results demonstrate that retro-orbital injection can be used for ^99mTc-MAG3 renal scintigraphy in mice. Therefore, in this study we assessed renal function in UUO mice using retro-orbital injections of ^99mTc-MAG3.

Renal scintigraphy was carried out in UUO and sham-operated mice at 0 (3 hours), 1, 3, and 6 days after surgery. Non-surgery mice were also imaged as a control. Figure 4 A-D shows TACs of UUO kidneys at each time point. As shown in Figure 4B, renal uptake of ^99mTc-MAG3 was remarkably reduced in UUO kidneys as early as day 1 post obstruction, as indicated by low total activity throughout the TACs, and it was further decreased as the disease progressed (Figure 4C and 4D). The TACs in UUO kidneys plateaued over time, indicating that ^99mTc-MAG3 is not excreted in these kidneys. Profound reduction of ^99mTc-MAG3 uptake was also demonstrated in UUO kidney by the time interval images of renal scintigraphy (Figure 5). Further, static renal scintigraphy demonstrated that excretion of ^99mTc-MAG3 to renal pelvis is highly limited in UUO kidneys, even at day 0 (3 hrs) post surgery (Figure 6); the finding indicates that TACs of UUO kidneys mostly demonstrate the deposition of ^99mTc-MAG3 in renal parenchyma. The contralateral kidneys in UUO mice (Figure 4 A-D) and kidneys in sham-operated mice (Figure 4E and 4F) showed similar TACs. Table 1 and Figure 7 show the parametric analysis of ^99mTc-MAG3 renal scintigraphy based on TACs obtained from UUO and sham operation mice at day 0, 1, 3, and 6 post surgery. The results of statistical analysis are shown in Table 2. As compared to sham-operated kidneys, SIU was remarkably (> 60%) decreased in UUO kidneys as early as day 1 post obstruction, while contralateral kidneys showed SIU values comparable to those in sham-operated kidneys (Figure 7A). It is noteworthy that the slope of the ascending phase (50s to peak, denoted Phase 2), which follows the initial linear uptake of ^99mTc-MAG3 (10s to 40s, denoted Phase 1), was progressively increased in contralateral kidneys, though it was not statistically significant against day 0 or sham operation kidneys (Figure 8). The plateau activity of TACs was also remarkably (~65%) reduced in UUO kidneys as early as day 1 post obstruction and it declined further as the disease progressed, while the peak activity in contralateral kidneys was comparable to those in sham-operated kidneys during the study period (Figure 7B). Further, the time-to-plateau (TTPt) in UUO kidneys largely exceeded the time-to-peak (TTP) values in sham-operated or contralateral kidneys (Figure 7C), suggesting that ^99mTc-MAG3 is slowly up-taken in UUO kidneys. The TTP was unaltered in contralateral kidneys as compared with sham operation kidneys, yet contralateral kidneys at UUO day 6 showed significantly shorter TTP than at UUO day 0, 1, and 3 (Figure 7C, Table 2). There was no significant difference in the peak:10min ratio, an indicator of tubular excretion rate [5, 6], between contralateral kidneys and sham-operated kidneys, though contralateral kidneys showed lower peak:10min ratios at day 0 and day 1, perhaps due to intravascular fluid shifts during surgical procedure (data not shown). No difference was observed in SIU, peak activity, TTP, and peak:10min ratio between sham-operated and non-surgery mice kidneys and between sham-operated and contralateral kidneys in sham mice.