Background
Significant renal mass reduction induced by subtotal 5/6 nephrectomy (Nx) is a model of chronic kidney disease (CKD), which involves deterioration of renal function due to loss of substantial number of nephrons and compensatory hypertrophy in the remnant kidney. In this model, the development of hypertension, proteinuria, and progressive renal fibrosis eventually leads to end-stage renal disease [
1‐
3]. The incidence of cardiovascular events in CKD is more frequent and severe compared to the normal population [
4,
5]. Cardiovascular complications including heart failure or ischemic heart disease represent the leading cause of death in CKD patients [
5,
6]. Amongst others, components of the renin angiotensin (Ang) system (RAS), particularly Ang II type 1 receptor (AT
1R), are known to contribute towards the detrimental effects on the hemodynamic and inflammatory events associated with the pathogenesis of the disease [
7‐
9]. The efficacy of anti-RAS therapies, such as AT
1R blockers (ARBs) and Ang converting enzyme inhibitors (ACEIs), in slowing progression of renal dysfunction, treating hypertension, and reducing cardiovascular complications confirms the important role of the AT
1R signaling pathway in these disorders [
3,
10,
11]. Numerous large clinical trials demonstrated the beneficial effects of RAS blockade on clinical symptoms and outcomes of patients with CKD; however, marked interindividual variability were detected in response to these treatments ranging from recognizable clinical benefit to non-detectable benefits or even serious adverse effects [
12,
13]. PET imaging of AT
1R may help to predict the response to anti-RAS therapy and personalize medicine.
Many organs contain components involved in the synthesis and actions of the RAS, which are largely independent of the RAS systemic components and not accessible to routine laboratory testing [
14,
15]. The presence of a distinct intrarenal RAS adds complexity in interpreting its role in the progression of CKD. In rodents, there are two isoforms of AT
1R identified in rodents: the AT
1AR and AT
1BR [
16,
17]. The AT
1AR represents the homologous form of the human AT
1R and is widely distributed on luminal membranes throughout the nephron segments [
18].
Using different methodologies, previous reports have presented contradictory results on the temporal role played by AT
1Rs in the progression of kidney diseases. The exact alterations in renal AT
1Rs are not entirely understood, with studies reporting decrease in cortex and medulla AT
1R messenger RNA (mRNA) levels in (30–40 %); [
19] no change; [
20] or increase (>70 %) [
9] in the receptor protein expression associated with CKD rodent models.
Non-invasive in vivo imaging of the AT
1R will allow for the identification of receptor expression abnormalities in CKD, will allow better understanding of the contribution of AT
1R to the development of the disease, and will aid to guide medical therapies for effective management of patients. [
18F]FPyKYNE-losartan is a novel PET imaging agent displaying high tissue uptake in the kidney cortex and outer medulla and binding selectivity for AT
1R over AT
2R [
21]. It binds with high affinity to renal AT
1Rs (
K
D of 49.4 nM) and has antagonistic efficacy with fourfold less potency reduction of blood pressure (ED
50 of 25.5 mg/kg) relative to losartan. [
18F]FPyKYNE-losartan PET imaging exhibited excellent reproducibility in pigs [
22]. The current work aims to evaluate the capability of using [
18F]FPyKYNE-losartan PET to study in vivo renal AT
1R changes in Nx animal model of CKD.
Discussion
Previous PET studies have explored in vivo renal AT
1R alterations induced by dietary sodium changes in rats [
30] and in a porcine model of renal ischemia [
31] but not in the hypertensive Nx rat model of CKD. The removal of one kidney and two thirds of the other in the 5/6 Nx rat model of CKD induces a substantial reduction in the number of functioning nephrons [
1,
2]. Subsequently, the remnant kidney size is excessively hypertrophied as a compensatory effect that may start initially as a true adaptive response but becomes maladaptive later in the course of the disease with deterioration of the renal function [
3,
19,
32]. In the current study, the presence of renal dysfunction is evident by the development of hypertension and increased plasma creatinine levels, whereas no overt albuminuria is observed in the Nx rats. Early albuminuria is caused mainly by mechanical damage to the glomerular cells leading to impaired selectivity of the glomerular capillary wall and excessive protein ultrafiltration [
33], so it may be speculated that this stage of the disease was not reached yet in our experiment. No change in kidney Ang II levels was detected in this study, even though an increase in the remnant kidney was anticipated due to local intrarenal RAS activation [
20,
32]. The reduction in plasma Ang II is most likely due to secondary suppression of systemic RAS as reported previously with this model [
20,
32].
There is a fair amount of data on the cardiac consequences secondary to renal failure established in this model [
9,
34]. Notably, LV hypertrophy is one of the common features reported during the different stages of CKD in animal models and patients [
35,
36]. Likewise, LV hypertrophy was observed in the Nx rats in our study. This result is probably attributed to prolonged pressure overload induced by hypertension and not necessarily due to activation of AT
1R in the heart [
4,
5]. It was not possible to visualize [
18F]FPyKYNE-losartan uptake in the heart due to the very low density of myocardial AT
1Rs compared to the kidney [
37]. Such low cardiac expression requires a very high specific activity of the injected tracer formulation (>7000 Ci/mmol) to prevent saturation of the AT
1Rs [
38].
Cortical RBF was not statistically different in remnant kidney compared to whole left kidney of shams. Previous groups reported an increase in RBF immediately after renal mass reduction that was normalized within 1 week post-surgery [
39,
40]. However, normal blood flow can be explained by the presence of intact renal autoregulation in the Nx rat kidneys [
9,
41].
The reduction in AT1R binding obtained with PET [18F]FPyKYNE-losartan in Nx rat kidneys was quantified using different parameters: DVs, SUVs, and KBR values. Physiologically, the Logan-derived DV values are the most appropriate indicators of protein expression and/or receptor-ligand binding potential (Bmax/Kd) for ligands that bind reversibly. However, due to technical limitations, no further corrections were applied for plasma input function, plasma protein binding, and non-specific binding which are implicit to the DV calculation. Hence, to enhance the validity of the measured DV values, PET findings were also represented using semiquantitative analysis (KBR and SUV), where an arterial input function is not indicated. The strong agreement between calculated SUV and DV encourages our confidence in the accuracy of the detected results.
In fact, our imaging data agrees with prior work displaying a decrease in AT
1R mRNA and protein expression and similar renal mass reduction at different timepoints [
19,
42]. Furthermore, Cao et al. reported that the reduction of AT
1R expression induced an imbalance in the relative proportions of AT
1 and AT
2 receptors that is consecutively implicated in progressive renal injury [
43]. The lower expression of AT
1R could be interpreted as a protective mechanism to avoid deleterious effects of hyperactive intrarenal RAS. On the other hand, there are studies that reported opposite effects with elevated AT
1R expression using Western blot following renal ablation [
9,
42]. AT
1R is implicated in most of the detrimental effects of CKD such as inflammation, renal fibrosis, and renal hypertrophy [
9]. A distinct speculation for the inconsistency in the AT
1R results can be due to the non-specificity of the commonly used AT
1R antibodies for in vitro assessment as demonstrated in two recent publications [
44,
45]. This uncertainty justifies the use of autoradiography in our study in addition to Western assays in assessment of AT
1R renal expression.
However, to be noted, in vitro autoradiography evaluates only the extent of membrane-bound receptor in non-viable tissue, whereas change in AT
1R density can be affected by internalization or turnover of the receptor. Activation of AT
1R signal transduction systems can occur within seconds through G
αq and IP
3 or within minutes to hours through MAP kinase and JAK/STAT systems [
46]. Moreover, AT
1Rs are endocytosed within 10 min after activation, with ~25 % recycled to plasma membrane and the remainder degraded in lysosomes [
47]. Consequently, the dynamic nature of AT
1R is a limitation for the accuracy of in vitro measurements and represents an added value for in vivo PET as an investigative tool to detect total receptor changes.
A non-invasive means for assessing renal AT
1R signaling at various stages of CKD in vivo would advance our understanding of the receptor abnormalities associated with progression of the disease and therapy response in patients. Measurements of a local tissue RAS component (such as AT
1Rs) in clinical or experimental CKD studies may be more predictive of the degree of renal injury compared to studies targeting the circulating RAS components. Nevertheless, it is important to note that AT
1R regulation is cell and tissue specific [
48], whereas a major drawback with PET imaging is its poor resolution (especially in rodents) to delineate specific cellular localization of [
18F]FPyKYNE-losartan accumulation within the kidney.
Competing interests
Dr Rob deKemp receives revenue shares from FlowQuant sales.
Authors’ contributions
Animal surgeries, PET imaging, echocardiography, SBP measurements, creatinine and albumin assays, westerns, autoradiography, and all data analysis described in this manuscript were conducted by BI, under supervision and guidance of JDS. RdK assisted with the application of kinetic modeling and quantification of PET data. TH conducted the synthesis of the novel radiotracer [18F]FPyKYNE-losartan. Some of the blood flow data was obtained from the MSc work of KM. RSB participated in the clinical implications and perspective on the AT1R imaging data. All authors read and approved the final manuscript.