**5.5. Metabolic activity (18F-FDG)**

<sup>18</sup>F-FDG is a daily routine tracer to assess regional glucose metabolism as a surrogate for met‐ abolic activity widely used for the PET-based routine detection of tumors, infection and in‐ flammation. The major energy source in leukocytes during the metabolic burst is glucose. Analogously, activated leukocytes highly accumulate 18F-FDG (in the same way they take up glucose but without further processing) which can be quantified by PET [42]. A clear limitation when using free 18F-FDG is that an increased uptake can be observed in any kind of cellular activation (high glycolytic activity). Hence, 18F-FDG is not a disease or target specific tracer.

Nevertheless, 18F-FDG is one of the few tracers successfully applied for the non-invasive de‐ tection of AR. Others have applied 18F-FDG in settings of lung, heart and liver transplantations. We have demonstrated very promising results for 18F-FDG-PET in diagnostics of renal AR [43;44]. Using a rat model of renal AR, 18F-FDG-PET performed well in terms of early, accurate detection and follow-up of AR [43] (Fig. 2). Using 18F-FDG, we discriminated AR non-inva‐ sively from important differential diagnoses like ATN or acute cyclosporine toxicity. More‐ over, therapy response monitoring by 18F-FDG might be useful to identify treatment unresponsive AR for earlier escalation of immunosuppressive regimen [44]. This might reduce graft damage by shortening AR episodes because at present (steroid) resistant rejection is di‐ agnosed lately [45].

One important issue of imaging of kidney AR with 18F-FDG is that it is eliminated with the urine in contrast to normal glucose. Thus, drainage of 18F-FDG into the renal pelvis must be taken care of when assessing 18F-FDG-uptake in the renal parenchyma. We avoided this prob‐ lem by using late acquisitions after 18F-FDG injection to reduce the instantaneous amount of tracer in the urine during the PET scan. Moreover, an impact of renal function on 18F-FDGuptake has to be excluded e.g. by renal fluoride clearance (a non-invasive measure of renal function) [46].

after surgery whereas graft hypoxia assessed in the long run is associated with clinical/subclin‐ ical rejection [53]. Therefore, assessment of hypoxia by targeting hypoxia (related gene prod‐ ucts), i.e. hypoxia inducible factors (HIF), might offer additional diagnostic information in

Non-Invasive Diagnosis of Acute Renal Allograft Rejection − Special Focus on Gamma Scintigraphy and Positron…

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Two major classes of hypoxia tracer, nitroimidazoles and *bis*(thiosemicarbazonato)copper(II) complexes, have been extensively investigated for measuring hypoxia. The applications of both tracer as well as several alternative reagents tested e.g., 18F-fluoroerythronitroimidazole

18F-FETNIM) and 18F-fluoroazomycin-arabinofuranoside (18F-FAZA), are summarized in a review recently published by Krohn *et al.* [54]. Until present and to the best of our knowledge, no study has been performed assessing hypoxia in renal AR by SPECT or PET so far. At least one has to evaluate if the SPECT and PET-based approaches are advantageous when compared

Apoptosis in AR is probably the result of different events occurring in AR. It may be a direct consequence of different cytokines discharged by leukocytes or directly provoked e.g. by CTLs. Within the inflammatory milieu of AR apoptosis might, among other factors, also be related to hypoxia, acidosis, or reactive oxygen species. Non-invasive detection of apoptosis in AR might be attractive because it may not serve for early detection of AR only, but also for mon‐ itoring of rejection kinetics and therapy response. Especially, early assessment of therapeutic success or failure is interesting to promptly adjust the therapeutic regimen. Likewise, quanti‐ fication of apoptosis might provide information regarding the extent of graft damage and therefore for its prognosis. However, small studies with different tracers targeting different steps in apoptosis have been performed in both, animal and man. A comprehensive review on detecting cell death *in vivo* has been recently published by us [55]. Two main operational strat‐ egies are followed. While imaging of caspases` activity using substrate-derived agents offers high selectivity, the detection of membrane phospholipid redistribution using extracellular agents has the advantage of high target density and accessibility [56]. We and others recently proposed different isatin analogues for 18F-labeling and detection of apoptosis [55]. However, studies detecting apoptosis in renal grafts using radiotracers for evaluation of their potential

A rather unspecific but reasonable approach is to simply determine graft function as a surro‐

Especially scintigraphic methods have been established for the assessment of renal function. Primarily, two types of imaging are common: static and dynamic. 99mTc-dimercaptosuccinic acid (DMSA) is the tracer used in static imaging allowing on the one hand identification of pathological conditions such as anatomical abnormalities or scarring, on the other hand accu‐ rate assessment of the differential function of the kidneys [57]. DMSA uptake correlates with the effective renal plasma flow, glomerular filtration rate, and creatinine clearance. Therefore,

subclinical or ambiguous cases of AR.

clinical value in AR have not been performed yet.

gate for stable function or (acute) graft affection.

**5.9. Imaging allograft function**

(

to BOLD MR.

**5.8. Apoptosis**

**Figure 2.** Representative PET-images of dynamic whole body acquisitions of a series of an allogeneically kidney trans‐ planted rat (POD 1 (A), 2 (B), 4 (C), and 7 (D), after tail vein injection of 18F-FDG. While the parenchyma (yellow circle) of renal allograft developing AR accumulates 18F-FDG with a maximum on post operative day (POD) 4, the native kid‐ ney (green circle) does not show any accumulation at any time. Please note that the renal pelvis can contain eliminat‐ ed 18F-FDG/18F-fluoride. Therefore, it has to be excluded from the measurements. Figure taken from [43]. Scale bar: percent injected dose

#### **5.6. Matrix metalloproteinases**

One step further, one cannot assess infiltrating leukocytes only but rather their tissue damag‐ ing activity by detection of activated matrix metalloproteinases (MMPs). Leukocyte-derived MMPs, like MMP-2 or MMP-9, were found to be active in AR [47;48]. Since MMP activity can be assessed using radiolabeled MMP-inhibitors in SPECT or PET this approach for detection of AR might be evaluated in future studies [49-52]. Maybe one can gather additional informa‐ tion regarding graft`s prognosis because MMPs are involved in tissue remodelling, too.

#### **5.7. Hypoxia**

Acute tissue inflammations regardless of their origin present with a unique and challenging microenvironment including hypoxia (low oxygen), anoxia (complete lack of oxygen), hypo‐ glycemia (low blood glucose), acidosis (high H+ concentration) and abundant free oxygen rad‐ icals. These conditions are characteristic features of inflamed tissues, along with the influx of leukocytes. In renal allografts, hypoxia and hypoxic adaptation are common within 2 weeks after surgery whereas graft hypoxia assessed in the long run is associated with clinical/subclin‐ ical rejection [53]. Therefore, assessment of hypoxia by targeting hypoxia (related gene prod‐ ucts), i.e. hypoxia inducible factors (HIF), might offer additional diagnostic information in subclinical or ambiguous cases of AR.

Two major classes of hypoxia tracer, nitroimidazoles and *bis*(thiosemicarbazonato)copper(II) complexes, have been extensively investigated for measuring hypoxia. The applications of both tracer as well as several alternative reagents tested e.g., 18F-fluoroerythronitroimidazole ( 18F-FETNIM) and 18F-fluoroazomycin-arabinofuranoside (18F-FAZA), are summarized in a review recently published by Krohn *et al.* [54]. Until present and to the best of our knowledge, no study has been performed assessing hypoxia in renal AR by SPECT or PET so far. At least one has to evaluate if the SPECT and PET-based approaches are advantageous when compared to BOLD MR.
