We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.
The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.
1. Donor Organ Recovery
2. Recipient Preparation and Transplantation
3. Imaging Allograft Rejection
Histology
During AR leukocytes, i.e. mainly T-lymphocytes are recruited into the transplant, whereas the severity of the rejection is reflected by the degree of inflammation. In the Periodic-Acid-Schiff (PAS) staining depicted here (Figure 1), the renal allograft shows significant histological signs of AR, namely glomerulitis, tubulitis, endothelialitis and graft infiltration (Figure 1, aTX POD4) (POD = postoperative day) while signs of rejection are absent in the native control kidney (Figure 1, CTR), Graft infiltrating cells are highly metabolically active cells which consume large amounts of glucose. However, if the latter is substituted with FDG, this will accumulate in the cells and can be measured and quantified by PET.
PET Images
Representative PET images of dynamic whole body acquisitions of a series of an allogeneically transplanted rat after tail vein injection of 30 MBq FDG (maximum a posterior MAP projection, 180 min p.i.) (Figure 2). A typical FDG distribution is found with distinct physiological accumulation in brain, heart, bone marrow and harderian glands. Moreover, free filtrated FDG accumulates in the urinary tract. In renal grafts undergoing AR the parenchyma (yellow circle, left kidney) highly accumulates FDG with a maximum on POD4, whereas the native kidney (green circle, right kidney) does not show any accumulation at all. Since the renal pelvis can contain free eliminated FDG, it was excluded from further measurements. Figure taken from 3.
Quantitative evaluation
For quantitative evaluation images were reconstructed, volumes of interest were traced manually around the kidneys according to 18F-fluoride perfusion and projected on the FDG images. After exclusion of the renal pelvis mean FDG uptake in the renal parenchyma was calculated by the ratio of total counts to volume (%ID ± SEM). Kidneys developing AR showed significantly increased FDG accumulation on POD4 (0.8 ± 0.06%) when compared to native controls (0.2 ± 0.02%) or syngeneically transplanted kidneys (0.37 ± 0.04%). Moreover, two major differential diagnoses of AR, namely acute tubule necrosis like in ischemia/reperfusion injury (IRI) (0.31 ± 0.02%) and acute calcineurin inhibitor toxicity (CSA) (0.16 ± 0.01%) did not show an elevated FDG accumulation and can therefore be distinguished from AR (Figure 3 taken from 3).
Figure 1. Histology. Signs of acute rejection, namely glomerulitis, tubulitis, endothelialitis, and graft infiltration, were found in the allograft group (aTX) and were completely absent in control kidneys (CTR).
Figure 2. FDG-PET Image. Representative PET-images of dynamic whole body acquisitions of a series of an allogeneically transplanted rat. In comparison to control kidneys (green circles) accumulates the parenchyma of renal allografts (yellow circles) FDG with a maximum on POD4. Since the renal pelvis can contain free eliminated FDG it was excluded from the measurements. Figure taken from 3.
Figure 3. Quantitative evaluation. Detection of acute rejection by measurement of the %ID of FDG. Renal allografts (aTX) exhibit significant higher FDG accumulations than control kidneys (CTR), syngeneically allografts (sTX), kidneys with acute tubule necrosis (ATN) or kidneys with acute calcineurin inhibitor toxicity (CSA) with a maximum on POD4 (aTX: 0.8 ± 0.06% CTR: 0.2 ± 0.02%, sTX: 0.37 ± 0.04%%, ATN: 0.31 ± 0.02%%, CSA: 0.16 ± 0.01%). Figure taken from 3.
FDG-PET imaging is a new option for the diagnosis of acute rejection. Because of its non-invasive and specific nature, FDG-PET is advantages in comparison to classical diagnostics by core needle biopsy. In contrast to the limited sample size of a biopsy, FDG-PET analysis the whole graft. Moreover, one can apply it to patients on anticoagulant therapy, and one can perform PET measures repetitively e.g. to monitor treatment efficiency 4. In addition, we already have shown that two major differential diagnosis of AR, namely acute tubule necrosis caused by ischemia reperfusion injury and acute calcineurin inhibitor toxicity, can be differentiated from AR using FDG-PET 3. Since FDG-PET imaging requires relatively low amounts of activity and imaging of either transplant patients or/and patients with impaired renal function is not associated with an increased risk of severe complications this approach can be easily transferred into daily clinical routine.
Nevertheless, one has to keep in mind that FDG is a rather non-specific tracer assessing regional metabolic activity. Thus, graft infection or tumors might lead to false positive results as well. Drainage of FDG into the renal pelvis might be a problem when assessing FDG uptake in the renal parenchyma. Therefore, we have chosen a late acquisition time three hours after injection to reduce tracer accumulation in the kidneys caused by renal excretion of FDG. In addition, the renal pelvis has to be carefully excluded when quantifying the renal FDG uptake. According to this protocol PET can be used to non-invasively detect AR, to differentiate it from ATN and CSA, and to perform serially investigations for follow-up or for evaluation of treatment efficiency 3, 4, 6. PET using 18F-fluoride is also useful to assess (split) renal function by calculation of the renal fluoride clearance as published before 5.
The allogeneic renal transplantation model using LBN F1 donor and Lewis recipient rats is an ideal model for investigation of acute cellular allograft rejection. In the absence of immunosuppression the allograft kidneys develop typical histological signs of AR according to the BANFF classification 7. Further, depending on the chosen modality (uni- vs. binephrectomized transplantation 3, 8-10) metabolic data can be evaluated as well to monitor allograft function. Due to the absence of immunosuppressive treatment, wound or systemic infections of the rats are extremely rare. Common surgical complications of this model include vessel stenosis, mostly seen at the insertion points of the graft vessels into the aorta or ICV of the recipient. This might cause ischemia or graft thrombosis. One can avoid this complication by using longer incisions in aorta and IVC. Sometimes uremia is found due to graft failure or ureter leakage caused by ureter necrosis or disconnection of the ureter from the bladder. If signs of uremia e.g. apathy, loss of appetite or spontaneous weight gain occur, the animals should be euthanized immediately.
The authors have nothing to disclose.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 656, Münster, Germany, projects C7 & C6) and the IZKF Münster (Core unit SmAP). The authors are grateful to Truc Van Le, Anne Kanzog, Ute Neugebauer, Wiebke Gottschlich and Roman Priebe for excellent technical assistance and to Daniel Burkert and Sven Fatum for producing radiotracers.
Equipment | |||
Mathieu Needle Holder – 14 cm | Fine Science Tools | 12010-14 | |
Castroviejo Micro Needle Holder – 9 cm | Fine Science Tools | 12061-01 | |
Surgical Scissors – Sharp_Blunt | Fine Science Tools | 14001-12 | |
Iris Scissors – ToughCut Straight 11.5 cm | Fine Science Tools | 14058-11 | |
Student Vannas Spring Scissors | Fine Science Tools | 91500-09 | |
Vannas Spring Scissors – 3 mm Blades | Fine Science Tools | 15000-00 | |
Student Tissue Forceps – 1×2 Teeth 12 cm | Fine Science Tools | 91121-12 | |
Dumont SS-45 Forceps – Inox Medical | Fine Science Tools | 11203-25 | |
Micro-Serrefine Clip Applicator with Lock | Fine Science Tools | 18056-14 | |
Micro-Serrefine 6 mm x 1 mm | Fine Science Tools | 18055-03 | |
Micro-Serrefine 4 mm x 0.75 mm | Fine Science Tools | 18055-04 | |
Reagent | |||
Isoflurane (e.g. Forene 100% v/v) | Abott | ||
cutane antiseptic (e.g. Octeniderm) | Schülke | ||
Povidone Iodine (e.g. Betaisodona) | Mundipharma | ||
ophthalmic ointment (e.g. Bepanthen) | Bayer | ||
Buprenorphin (e.g. Temgesic) | RB Pharmaceuticals | ||
HTK perfusion solution (e.g. CUSTODIOL HTK) | Dr. Franz Köhler Chemie | ||
surgical thread Mersilene 0 | Ethicon | EH6665E | |
surgical thread Mersilene 4-0 | Ethicon | EH6732H | |
surgical thread Prolene 6-0 | Ethicon | 8697H | |
surgical thread Ethilon 9-0 | Ethicon | 2809G | |
surgical silk 5-0 | Vömel | 14739 | |
Canula (e.g. Microlance 3, 27G ¾) | BD | 302200 |