Animals and experimental design
All animal experiments were conducted in accordance with institutional guidelines and approved by the Administrative Panel on Laboratory Animal Care (Government of Upper Bavaria, Germany). We used non-tumour bearing female Lewis rats (Charles River Laboratories, Sulzfeld, Germany), aged 10 weeks, with a median weight of 207 g, which were fed a standard diet and given free access to water. The body weight of all animals was monitored weekly. Animals were divided into four groups. Group 1 (n = 15) received placebo, group 2 (n = 17) everolimus (5 mg/kg body weight once weekly), group 3 (n = 14) a combination of placebo (once weekly) and [177Lu]Lu-DOTA-TATE (single injection at the start of the study, mean 200 MBq, range 191–207 MBq) and group 4 (n = 16) a combined treatment with everolimus (5 mg/kg weekly) and a single injection of [177Lu]Lu-DOTA-TATE at the start ot the study (mean 200 MBq; range 195–212 MBq). Based on the experience of Pool et al., the administered activity of 200 MBq [177Lu]Lu-DOTA-TATE represents a trade-off between low and high dose therapy and a potential curative dose after a single injection [11]. Renal function was monitored weekly (respectively every 14 days after week 8) by determination of creatinine and blood urea nitrogen (BUN) in the blood serum by drawing approximately 0.5 ml blood from a tail vein. At the end of the observation period, blood samples from the heart were collected to assess the full blood count. Furthermore, renal function of the rats was evaluated with serial [99mTc]Tc-MAG3-scintigraphies in the remaining half of rats. A baseline scan was performed in a group of 21 randomly chosen, otherwise untreated rats 1 week before the start of the actual treatment. Control MAG3 scans were performed in all animals in the four groups (group 1: n = 7, group 2: n = 7, group 3: n = 6, group 4: n = 8) 1, 6, 11 and 16 weeks after the start of the treatment. Laboratory studies (n = 8 in groups 1, 3 and 4; n = 10 in group 2) and renal scintigraphies were performed in different animals of the same group. All animals were euthanized 16 weeks after the start of the treatment, and the kidneys were prepared for the histopathological examination. No animal had to be euthanized due to severe toxicity prior to the endpoint of 16 weeks post treatment.
Laboratory chemical analysis
Creatinine and BUN levels in the serum were quantified to monitor kidney function. A total blood count was performed right before euthanasia of animals at the end of the study. All laboratory analyses were performed according to the manufacturer’s protocols and standardized methods at the Institute of Laboratory Medicine of the Medical Centre of the University of Munich. Blood was not diluted. Serum creatinine and BUN concentrations were measured using an Olympus AU5400 analyser (Beckman-Coulter) using the creatinine reagent OSR6178 and the urea reagent ORS6578. Blood count analysis was performed using an XN-2000 analyser (Sysmex, Kobe, Japan). All analyses were performed according to the manufacturer’s protocols.
Pharmaceuticals and radiopharmaceuticals
Everolimus (formerly known as RAD001) and placebo were kindly provided by Novartis Pharmaceuticals (Basel, Switzerland). We applied a weekly dose of 5 mg/kg body weight chosen in accordance with previously published data for single agent treatment [12]. The pharmaceuticals were freshly prepared from the pre-concentrate once weekly right before oral gavage. In accordance with the manufacturer’s manual, the everolimus pre-concentrate was diluted with 5% glucose solution to a concentration of 2 mg/ml corresponding to an administered volume of ~ 0.5 ml. Equivalent amounts of pre-concentrate and glucose solution were used for the preparation of the placebo solution. 99mTc-mercaptoacetyltriglycine was purchased from Covidien, Neustadt/Donau, Germany, and prepared according to the manufacturer’s manual. No carrier added 177Lu was obtained from Isotope Technologies Garching GmbH (Garching, Germany). DOTA0,TYR3-octreotate was obtained from ABX advanced biochemical compounds (Dresden, Germany). Radiolabeling was performed using 125 μg DOTA0,TYR3-octreotate according to a previously described protocol [13]. Quality control was performed using thin layer chromatography and HPLC. Radiochemical yield 99.9% and purity > 99.5% (molar activity GBq/mol). All radiopharmaceuticals were administered via the tail vein (with an administered volume of ~ 0.5 ml).
Renal scintigraphy
[99mTc]Tc-MAG3-scintigraphy was performed as described in previously published protocols [14,15,16,17]. Inhalational anaesthesia with 2.0% of isoflurane in pure oxygen was induced and maintained with a concentration of 1.5%. Rats received a standard dose of [99mTc]Tc-MAG3 (50 MBq) solved in 0.3 ml of sterile saline as a bolus via tail vein. Whole body scintigraphic recordings were initiated at the moment of tracer administration. One head of a triple-headed gamma camera (Philips–former Picker–Prism 3000 XP, Cleveland, USA) equipped with a LEHR collimator was on hand. The dynamic planar acquisitions consisted of 420 frames of 5 s each to a total of 35 min. For the baseline scans, 240 frames (20 min) were acquired to reduce the duration of anaesthesia.
In order to analyse generated data sets, the Hermes Dynamic Study display software V4.0 was used (Hermes Gold V2.10, Hermes Medical Solutions, Stockholm/London). Standardized regions of interest (ROI) was drawn for the whole body, both the kidneys, peri-renal background reference regions, the bladder, blood pool in the heart and the site of injection [15]. Further, dynamic data sets of the ROIs were used to create renograms using Microsoft Excel, which depicted the proportion of the kidney activity corrected for the background regions and the whole-body activity corrected for the injection site. The baseline renograms were extrapolated to 35 min using a monoexponential fit of the excretion phase. Eventually, the parameters ‘time to peak’, ‘peak’, ‘IA10min’ and ‘Delta10min’ were extracted from the renograms.
Furthermore, the fractional uptake rate (FUR) was calculated to assess renal clearance from the renograms [16]. FUR is defined as the fractional uptake of a tracer in the blood by an organ per time unit and can be calculated in the following way: FUR = P(0) × (kl+kr)/[IA]. P(0) was obtained by extrapolating backwards, using a mono-exponential fit of P(t). The figures kl and kr are the slopes of the linear uptake (LU) segment of the Patlak-Rutland (PR) plots for the left and right kidneys [17].
Histopathological analysis
After the kidneys were fixed in 10% neutral-buffered formalin solution, they were dehydrated under standard conditions and embedded in paraffin. All blocks were cut into 2 μm slices. Selected slices were stained with Periodic acid–Schiff (PAS), adjacent ones with Haematoxylin-Eosin (HE) according to standard protocols. Subsequently, renal damage was classified according to Rolleman’s grading scale using a renal damage score (RDS) ranging from grade 0 (no damage) to grade 4 (severe damage) [18, 19]. Briefly, evaluation criteria included the following:
Grade 1—inflammatory infiltrate in the glomeruli, little dilatation of tubules; no basal membrane thickening or protein cylinders
Grade 2—same criteria as for grade 1, however in addition rough protein staining, more pronounced dilation of tubules, basal membrane thickening and mitotic activity; very little protein cylinders in tubules
Grade 3—same criteria as for grade 2, however additional shrinkage in a small number of glomeruli, smaller vascular lumina flat or lost tubular epithelium, strong tubule dilatation and more pronounced basal membrane thickening; more protein cylinders
Grade 4—same criteria as for grade 3, however increased shrinkage of glomeruli leading to optical emptiness; strongly dilated tubules with massive protein cylinders and signs of peripheral fibrosis
The findings of the histopathological examination were recorded using the Excel sheet.
Statistical analysis
Data are expressed as the means of the treatment groups and the corresponding 95% confidence intervals. A p value of p < 0.05 was considered as statistically significant. Normality and homogeneity of variance were tested using the Shapiro-Wilk test and Levene’s test. To adjust for multiple testing, two-way analysis of variance (ANOVA) was carried out for parameters measured only once at the end of the observation period. When normality and/or homogeneity requirements were not met, the Scheirer-Ray-Hare (SRH) test was used, with the administration of everolimus or placebo as one and the treatment with or without [177Lu]Lu-DOTA-TATE as the second factor in both cases. For repeatedly measured parameters from the blood sampling, the ANOVA or SRH test was applied for the individual differences between the first and last measurement. By covering all events of a certain parameter (in our case the values of all animals in all our groups rather than only the animals of two specific groups), the validity of the tests used is increased. Moreover, the added value of ANOVA and SRH lies in the evaluation of an over-additive or synergistic effect by analysing the impact of a combination of PRRT and everolimus. Statistical analysis of scintigraphy results was performed after obtaining and averaging baseline parameters. Means at baseline were considered as the population standard. T-tests were conducted versus the population standard for the average of each group in the follow-up scintigraphies. Pearson’s chi-squared test was used to test for differences among the ordinally scaled values of the histological grading.