Value of [68Ga]Ga-FAPI-04 imaging in the diagnosis of renal fibrosis

Renal fibrosis is a pathological state in the progression of chronic kidney disease. Early detection and treatment are vital to prolonging patient survival. Renal puncture examination is the gold standard for renal fibrosis, but it has several limitations. This study aims to evaluate the diagnostic performance of a novel PET radiotracer, [68Ga]Ga-fibroblast activation protein inhibitor (FAPI)-04, which specifically images fibroblast activation protein (FAP) expression for renal fibrosis. All patients underwent renal puncture before receiving [68Ga]Ga-FAPI-04 PET/CT imaging. They then underwent [68Ga]Ga-FAPI-04 PET/CT and immunochemistry examinations. The data obtained were analyzed. The [68Ga]Ga-FAPI-04 PET/CT examination results demonstrated that almost all patients (12/13) exhibited increased radiotracer uptake. The maximum standardized uptake value (SUVmax) in patients with mild, moderate, and severe fibrosis was 3.92 ± 1.50, 5.98 ± 1.6, and 7.67 ± 2.23, respectively. Compared with renal puncture examination, non-invasive imaging of FAP expression through [68Ga]Ga-FAPI-04 PET/CT quickly demonstrates bilateral kidney conditions with high sensitivity. [68Ga]Ga-FAPI-04 PET/CT can facilitate the evaluation of disease progression, diagnosis, and the development of a treatment plan.


Introduction
Renal fibrosis refers to the pathological changes caused by the deposition of extracellular matrix in the kidney, including glomerulosclerosis, renal interstitial fibrosis, and arteriosclerosis [1,2]. It does not refer to a specific disease but rather to a pathological state in the progression of almost all chronic kidney diseases (CKDs), which often indicates an irreversible decline in renal function. Renal fibrosis is closely related to patient prognosis. As renal fibrosis progresses, functional nephrons gradually decrease, eventually progressing to endstage renal disease (ESRD) [3,4]. Due to a lack of reliable, easy assessment methods, it is difficult to precisely determine renal fibrosis prevalence. However, given the 10.8% Yue Zhou and Xin Yang contributed equally to this work.
This article is part of the Topical Collection on Radiopharmacy * Yue Chen chenyue5523@126.com Santao Ou ousantao@163.com prevalence of CKD, it can be inferred that many patients have renal fibrosis [5].
Fibroblast activation protein (FAP) is a member of the cell surface dipeptidyl peptidase (DPP) family of serine proteases. FAP is involved in inflammatory and fibrotic tissues [12][13][14][15][16]. Kidney damage can cause inflammatory cell infiltration and the myofibroblasts a large number of extracellular matrix components produced by, which in turn lead to renal fibrosis [17][18][19][20]. The radiolabeled FAP inhibitor [ 68 Ga]Ga-FAPI-04 was developed as a radiotracer for PET/CT imaging. At present, its clinical value for renal fibrosis has not been systematically studied. This research aims to improve the detection rate of renal fibrosis through non-invasive imaging technology and facilitate clinical diagnosis and treatment.
Patients, materials, and methods Patients A total of 13 patients who had undergone renal puncture at the Affiliated Hospital of Southwest Medical University between January and October of 2020 were enrolled in the study. Hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining showed that the patients' kidneys had varying degrees of renal fibrosis. The study excluded all patients with general health conditions affecting cardiopulmonary function/mental status and allergies to alcohol. This study was approved by the Ethics Committee of the First Affiliated Hospital of Southwest Medical University and followed the 1964 Helsinki Declaration and its subsequent amendments to the ethical standards. All patients signed written informed consent forms.
[ 68 Ga]Ga-FAPI-04 was provided based on compassionate use. All patients underwent PET/CT examinations, and immunochemical examinations were performed on the kidney tissues collected before patients were enrolled in the study (see Table 1 for patients' information). The average age of the patients was 42.0 ± 17.0 (18-69 years). All patients showed pathological findings (HE and PAS staining) of renal fibrosis. Five patients had mild fibrosis, five had moderate fibrosis, and three had severe fibrosis. The evaluation parameters (including glomerulosclerosis, renal tubular atrophy, interstitial inflammation, and fibrosis) corresponded to the three grades I, II, and III, respectively, equivalent to affected proportions of <25%, 25-50%, and > 50%.
We retrospectively analyzed nine patients who underwent [ 68 Ga]Ga-FAPI-04 examination in the Department of Nuclear Medicine of the Affiliated Hospital of Southwest Medical University whose kidneys were not detected with abnormal radiotracer uptake as a normal control group.

Preparation of [ 68 Ga]Ga-FAPI-04
We purchased the precursor FAPI-04 from MCE (MedChemExpress, USA) with a purity grade of 98% and a mass of 872.91. FAPI-04 radiolabeling was performed according to the following protocol: 50 μg of FAPI-04 was dissolved in 1 mL of sodium acetate solution (0.25 M) and added 4 mL 68 Ga-solution (1.7 GBq) to a pH of 3.3-3.6. The reaction was heated at 80°C for 10 min and the product was purified by using a Sep-pak 18 C column. It was then eluted with 1 mL of 50% ethanol and 4 mL of saline. Quality control was performed by radio-HPLC on an 18 C reverse phase column with a gradient elution of either H 2 O with 0.1% TFA (solvent A) or CH 3 CN with 0.1% TFA (solvent B). The mobile phase conditions were 0-50 min: 10-90% B, 1 mL/min. The radiochemical purity was over 98%.

Imaging and image analysis
Imaging was performed after the patient was in the Department of Nephrology, Affiliated Hospital of Southwest Medical University. Each patient provided a detailed medical history and underwent a physical examination before imaging. The intravenous radiotracer dose was 1.85-2.59 MBq/kg, and imaging was performed 50-60 min after radiotracer injection. All patients were required to urinate as much as possible for imaging preparations, which reduces the influence of the residual radiotracer in the renal pelvis and calyces. Some patients with poor renal function (GFR < 60 mL/min, urine volume < 1000 mL/24 h) were given diuretics (Furosemide, 0.57 mg/kg). The scope of the whole-body inspection was from the base of the skull to the base of the thigh, using five to six beds (3 min/bed). The matrix was 128 × 128, the PET layer thickness was 3 mm, and all PET images were reconstructed iteratively. All of the above inspection procedures were communicated to patients before obtaining their written informed consent.
The [ 68 Ga]Ga-FAPI-04 PET/CT image interpretation was based on visual and semi-directional analysis and was evaluated by two experienced nuclear medicine doctors. The mean standardized uptake value (SUVmean) of a round sphere with a diameter of 2 cm was selected from the liver as the activity background, and the SUVmax of the renal parenchyma was divided by this SUVmean to calculate target-to-background ratio (TBR). VOIs were located in the kidney parenchyma with increased radiotracer uptake [21], and the renal pelvis and calyces were avoided. The ROI diameter used by the kidney was 1 cm. Nuclear medicine and nephrology physicians checked the patient's general condition (mental state/ blood pressure/heart-rate/body temperature) until 120 min after radiotracer injection and were required to report any abnormalities.

Immunochemistry
The kidney tissue was stained with an antibody against fibroblast activation protein-α (FAPα), treated with formalin, and then embedded in paraffin. All kidney tissues were obtained from the archives of the Department of Pathology, Affiliated Hospital of Southwest Medical University. The immunohistochemical image was evaluated by the scoring system adopted by Henry et al., in which kidney tissue is evaluated as 0 (no FAP immunostaining), 1+ (< 10% of stromal cells showing positive FAP staining), 2+ (10-50% of stromal cells showing positive FAP staining), and 3+ (> 50% of stromal cells showing positive FAP staining).

Statistical analysis
SPSS software (version 26.0; IBM, Armonk, NY) was used for statistical evaluation, and Graphpad8.0 was used for graphing. Measurement data were expressed as mean ± standard deviation. The Kruskal-Wallis nonparametric rank-sum test was used to compare the different degrees of renal fibrosis between groups. The Mann-Whitney U test was used to compare between sexes. *P < 0.05 represents a comparison of the control group; #P < 0.05 represents a comparison of the treatment group.

Diagnostic performance of [ 68 Ga]Ga-FAPI-04 PET/CT in primary tumors
In this study, significant radiotracer uptake was detected in most patients (12/13). As the patients' HE and PAS staining showed the degree of renal fibrosis gradually increasing, patients' SUVmax, serum creatinine (Scr), and TBR gradually increased and their glomerular filtration rate (GFR) gradually decreased. Patient indicators for different grades of renal fibrosis were compared by mean ± standard deviation. The renal SUVmax of healthy individuals (2.35 ± 0.55) was statistically significantly lower than that of patients with renal fibrosis grade II (5.98 ± 1.67) and III (7.67 ± 2.23) (P < 0.05). Meanwhile, the TBR of healthy individuals (3.30 ± 0.92) was statistically significantly lower than that of patients with renal fibrosis grade I (7.07 ± 2.84), II (8.35 ± 1.40), and III (11.74 ± 1.60) (P < 0.05). The GFR of patients with renal fibrosis grade I (86.26 ± 23.58) was statistically significantly higher than that of patients with renal fibrosis grade III (17.23 ± 12.49) (P < 0.05). However, the difference between patients in other groups was not statistically significant (P > 0.05). Figures 1, 2, 3, and 4 show some patient examination images, and Tables 2 and 3 and Fig. 5 show the study's statistical results.

Immunochemistry
Through immunohistochemical staining of antibodies specific for FAPα, FAP expression in renal interstitial cells was found in 10 patients (76.9%) and FAP expression in glomerular cells was found in four patients (

Incidental findings
A diffusely increased radiotracer uptake (SUVmax = 11.7) was found in the breast of one young female patient, which may have been related to the patient's dense glands or changing hormone levels [22,23]. Two patients' bilateral erector spinae also had increased radiotracer uptake. After questioning the two patients, we determined that they were employed in agriculture and as a bus driver, which may have damaged their erector spinae. They were diagnosed with erector spinae strain.

Adverse events
All patients tolerated the [ 68 Ga]Ga-FAPI-04 PET/CT examination well. There were no signs of drug-related pharmacological effects or physiological reactions. All patients' vital signs (blood pressure/heart-rate/body temperature) were kept within the normal range before, during, and after the examination. No patients reported any abnormalities.

Comparison with other non-invasive imaging examinations and suitability
At present, the main non-invasive imaging examinations for renal fibrosis are magnetic resonance, ultrasound, and enhanced CT. There is a lack of consensus regarding MRI's detection of renal fibrosis due to conflicting reports, which affects reliability [24]. Ultrasound examination of renal fibrosis has low specificity and is easily affected by factors such as the doctor's subjective judgment, examination preparation, and abdominal effusion [25,26]. Enhanced CT may damage the patient's kidneys due to the use of contrast agents and intravenous hydration [27]. [ 68 Ga]Ga-FAPI-04 PET/CT may be suitable for patients with renal fibrosis, including abnormal blood coagulation function, renal parenchymal atrophy, acute renal fibrosis, and negative puncture tests.

Discussion
To date, no serological indicators have been found that specifically reflect the degree of renal fibrosis. Blood lysyl oxidase (LOX), human epididymis protein 4 (HE4), and pentraxin-2 may potentially assess renal fibrosis, but are easily affected by other fibrotic diseases (liver fibrosis, pulmonary fibrosis, etc.) [28][29][30]. Non-invasive imaging examinations, such as B-ultrasound, indirectly reflect renal fibrosis through renal anatomical changes, making it difficult to determine the degree of renal fibrosis early and accurately. Due to the potential impact of contrast agents on renal function, enhanced CT is also unsuitable for evaluating renal fibrosis, especially  for ESRD patients. The current imaging methods for assessing the degree of renal fibrosis are limited [31]. At present, kidney tissue biopsy is the main method of accurately assessing the presence and severity of renal fibrosis. Although reliable, it has some contraindications and may cause complications, which hinders its wide clinical application. It can only reflect fibrosis of the kidney tissue at the puncture site and not the overall degree of fibrosis [32].   examination (g, h) found FAP in the renal interstitium, while no FAP was found in the globular tissue. The pathological results were consistent with the PET/CT results. In this case, the patient's bilateral erector spinae radiotracer uptake was slightly increased and the SUVmax was 3.0. Upon questioning, we found that the patient had a long-term occupational history of manual agriculture. The patient was diagnosed with erector spinae strain Therefore, it is vital to find other non-invasive methods with better specificity and sensitivity to accurately assess the degree of renal fibrosis.
In this study, [ 68 Ga]Ga-FAPI-04 was used to detect renal fibrosis. It proved to be a promising new imaging method, with a higher uptake of radiotracer found in almost all patients (12/13). It was easier to distinguish the radiotracer with higher uptake of renal parenchyma from the background because of the high average values of TBR and SUVmax. We chose the liver as the background organ because the kidneys typically   ) found FAP in the renal interstitium. Almost no FAP was found in the glomerulus, which was consistent with the PET/CT results take up radiotracer diffusely, and it is difficult to choose normal kidney tissue as the background. Among the patients included in our study, a small number have specific uptake of radioactivity in the back muscles. In addition, patients with chronic kidney disease often suffer from protein loss from the whole body, leading to muscle edema and thinning. Therefore, we did not choose muscle tissue as the background. We did not select the skeletal system as the background as renal osteopathy causes bone changes.
In the included patients, liver uptake was uniform and was not affected by kidney disease, so we chose the liver as the background. The SUVmax and TBR of most patients correlated with the pathology of kidney tissue. [ 68 Ga]Ga-FAPIs PET/CT examination may enable diagnosis of renal fibrosis without a biopsy. Clinicians can instruct the kidney biopsy site for patients with focal renal fibrosis. [ 68 Ga]Ga-FAPIs PET/CT can also detect early renal fibrosis. Separate examinations before and after nephropathy treatment may also evaluate the effectiveness of clinical treatment methods.
This study has some limitations, including the fact that a small number of patients (2/13) had some residual radiotracer in the renal pelvis and calyces, which may have affected image quality. Also, the small number of study subjects and the unequal number of patients with various types of renal fibrosis limited the statistical significance. In the future, a larger patient cohort is needed to evaluate [ 68 Ga]Ga-FAPI PET/CT in renal fibrosis. In summary, compared with traditional renal puncture examinations, [ 68 Ga]Ga-FAPIs PET/CT can quickly show the histology of renal fibrosis. In patients for whom renal puncture is unsuitable, [ 68 Ga]Ga-FAPIs PET/CT scans can be used to determine rapid and effective treatment plans for improved results.

Conclusion
The results of this preliminary study indicate that radiolabeled FAPI can be used to image renal fibrosis. The imaging quality of [ 68 Ga]Ga-FAPI-04 PET/CT scans in patients with moderate-to-severe renal fibrosis may be superior to other examination methods, but additional clinical trials are needed for further evaluation.

Declarations
Ethics approval and consent to participate All procedures involving human participants were performed in accordance with the ethical standards of the institutional committee, as well as the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This article does not contain any animal experiments. Informed consent was obtained from all participants included in the study.
Consent for publication Informed consent was obtained from all participants included in the study.

Conflict of interest
The authors declare no competing interests.