Most physiologic effects of the renin angiotensin system (RAS) are mediated via the angiotensin (Ang) type 1 receptor (AT 1 R). The 18 FFPyKYNE derivative of the clinically used AT 1 R blocker losartan exhibits high binding selectivity for kidney AT 1 R and rapid metabolism in rats. The aim of this study was to further assess the binding profile of this novel PET agent for imaging AT 1 R in rats and pigs. Methods: In vitro binding assays were performed with 18 F-FPyKYNElosartan in rat kidneys. Male Sprague-Dawley rats were used to assess dosimetry, antagonistic efficacy via blood pressure measurements, and presence of labeled metabolites in kidneys. Testretest PET imaging, blocking with AT 1 R antagonist candesartan (10 mg/kg), and plasma metabolism analysis were performed in female Yorkshire pigs. Results: 18 F-FPyKYNE-losartan bound with high affinity (dissociation constant of 49.4 ± 18.0 nM and maximal binding of 348 ± 112 fmol/mm 2 ) to rat kidney AT 1 R. It bound strongly to plasma proteins in rats (97%), and its labeled metabolites displayed minimal interference on renal AT 1 R binding. FPyKYNE-losartan fully antagonized the Ang II pressor effect, albeit with 4-fold potency reduction (the effective dose inhibiting 50% of the Ang II-induced maximal pressor response of 25.5 mg/kg) relative to losartan. PET imaging exhibited high kidney-to-blood contrast and slow renal clearance, with an SUV of 14.1 ± 6.2. Excellent reproducibility was observed in the calculated test-retest variability (7.2% ± 0.75%). Only hydrophilic-labeled metabolites were present in plasma samples, and renal retention was reduced (−60%) at 10-15 min after blockade with candesartan. Conclusion: 18 F-FPyKYNE-losartan has a favorable binding profile and displays high potential for translational work in humans as an AT 1 R PET imaging agent.
ACE inhibitors are considered first line of treatment in patients with many forms of chronic kidney disease (CKD). Other antihypertensives such as calcium channel blockers achieve similar therapeutic effectiveness in attenuating hypertension-related renal damage progression. Our objective was to explore the value of positron emission tomography (PET) imaging of renal AT1 receptor (AT1R) to guide therapy in the 5/6 subtotal-nephrectomy (Nx) rat model of CKD. Ten weeks after Nx, Sprague-Dawley rats were administered 10mg/kg/d enalapril (NxE), 30mg/kg/d diltiazem (NxD) or left untreated (Nx) for an additional 8–10 weeks. Kidney AT1R expression was assessed using in vivo [18F]fluoropyridine-losartan PET and in vitro autoradiography. Compared to shams, Nx rats exhibited higher systolic blood pressure that was reduced by both enalapril and diltiazem. At 18–20 weeks, plasma creatinine and albuminuria were significantly increased in Nx, reduced to sham levels in NxE, but enhanced in NxD rats. Enalapril treatment decreased kidney angiotensin II whereas diltiazem induced significant elevations in plasma and kidney levels. Reduced PET renal AT1R levels in Nx were normalized by enalapril but not diltiazem, and results were supported by autoradiography. Reduction of renal blood flow in Nx was restored by enalapril, while no difference was observed in myocardial blood flow amongst groups. Enhanced left ventricle mass in Nx was not reversed by enalapril but was augmented with diltiazem. Stroke volume was diminished in untreated Nx compared to shams and restored with both therapies. [18F]Fluoropyridine-Losartan PET allowed in vivo quantification of kidney AT1R changes associated with progression of CKD and with various pharmacotherapies.
BackgroundSignificant renal mass reduction induced by 5/6 subtotal nephrectomy (Nx) is associated with a chain of events that culminates in hypertension and chronic kidney disease (CKD). Numerous studies have provided evidence for the role of angiotensin (Ang) II type 1 receptor (AT1R) in the promotion and progression of the disease; however, conflicting results were reported on intrarenal AT1R levels in CKD models.MethodsMale Sprague-Dawley rats (n = 26) underwent Nx or sham operations. Animals were scanned at 8–10 weeks post-surgery with PET using the novel AT1R radioligand [18F]FPyKYNE-losartan. Radioligand binding was quantified by kidney-to-blood ratio (KBR), standard uptake value (SUV), and distribution volume (DV). After sacrifice, plasma and kidney Ang II levels were measured. Western blot and 125I-[Sar1, Ile8]Ang II autoradiography were performed to assess AT1R expression.ResultsAt 8–10 weeks post-surgery, Nx rats developed hypertension, elevated plasma creatinine levels, left ventricle hypertrophy, increased myocardial blood flow (MBF), and reduced Ang II levels compared to shams. PET measurements displayed significant decrease in KBR (29 %), SUV (24 %), and DV (22 %) induced by Nx (p < 0.05), and these findings were confirmed by in vitro assays.ConclusionsReduced renal AT1Rs in hypertensive rats measured with [18F]FPyKYNE-losartan PET at 8–10 weeks following Nx support further use of this non-invasive approach in longitudinal studies to better understand the AT1R role in CKD progression.
There is accumulating evidence suggesting that depression is a risk factor for cardiovascular diseases. This study aimed to examine the hypothesis that the proinflammatory cytokine TNF-α would partially explain the link between depression and atherosclerotic endothelial changes. Rats were distributed among 6 groups: (i) control group; (ii) group subjected to chronic mild stress (CMS); (iii) group fed a cholesterol-cholic acid-thiouracil (CCT diet); and (iv) CMS group fed the CCT diet and treated with the vehicle for 8 weeks. The last 2 groups were subjected to CMS-CCT and received thalidomide (THAL) or imipramine (IMIP). Rats were assessed behaviorally (sucrose preference, open field, and forced-swimming tests). TNF-α protein was assessed from the serum, aorta, and liver. Aortic TNF-α gene expression (assessed using RT-PCR), serum lipid profile, and insulin levels were measured. Endothelial function was assessed in isolated aortic rings. The THAL and IMIP groups showed ameliorated CMS-CCT-related behavioral changes. CMS-CCT-induced metabolic and endothelial dysfunctions were improved in the THAL group but were worsened in the IMIP group. RT-PCR showed a significant reduction of aortic TNF-α mRNA expression in the THAL and IMIP treatment groups. These data paralleled the findings for aortic immunohistochemistry. The THAL group, but not the IMIP group, showed improved CMS-CCT-induced changes in the vascular reactivity of the aortic rings. Thus, TNF-α provides a target link between depression, metabolic syndrome, and endothelial dysfunction. This could open a new therapeutic approach to address the comorbidities of depression.
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