Angiotensin type 1 receptor resistance to blockade in the opossum proximal tubule cell due to variations in the binding pocket

Abstract

Blockade of the angiotensin (ANG) II receptor type 1 (AT(1)R) with angiotensin receptor blockers (ARBs) is widely used in the treatment of hypertension. However, ARBs are variably effective in reducing blood pressure, likely due, in part, to polymorphisms in the ARB binding pocket of the AT(1)R. Therefore, we need a better understanding of variations/polymorphisms that alter binding of ARBs in heterogeneous patient populations. The opossum proximal tubule cell (OKP) line is commonly used in research to evaluate renal sodium handling and therefore blood pressure. Investigating this issue, we found natural sequence variations in the opossum AT(1)R paralleling those observed in the human AT(1)R. Therefore, we posited that these sequence variations may explain ARB resistance. We demonstrate that OKP cells express AT(1)R mRNA, bind (125)I-labeled ANG II, and exhibit ANG II-induced phosphorylation of Jak2. However, Jak2 phosphorylation is not inhibited by five different ARBs commonly used to treat hypertension. Additionally, nonradioactive ANG II competes (125)I-ANG II efficiently, whereas a 10-fold molar excess of olmesartan and the ANG II receptor type 2 blocker PD-123319 is unable to block (125)I-ANG II binding. In contrast, ANG II binding to OKP cells stably expressing rat AT(1A)Rs, which have a conserved AT(1)R-binding pocket with human AT(1)R, is efficiently inhibited by olmesartan. A novel observation was that resistance to ARB binding to opossum AT(1)Rs correlates with variations from the human receptor at positions 108, 163, 192, and 198 within the ARB-binding pocket. These observations highlight the potential utility of evaluating AT(1)R polymorphisms within the ARB-binding pocket in various hypertensive populations.

In silico prediction of ARB resistance: A first step in creating personalized ARB therapy

“…Since Olmesartan was crystalized in the AT 1 R (PDB: 4ZUD) [ 7 ], which served as our starting structure, and was the focus of our previous studies [ 20 ], we first docked Olmesartan back into the apo-AT 1 R model described above ( S1 File ). Three docking programs were utilized: AutoDock 4.2 ( Fig 4A ), AutoDock Vina ( Fig 4B ), and MOE ( Fig 4C ).…”

Estrogen Metabolite 2-Methoxyestradiol Attenuates Blood Pressure in Hypertensive Rats by Downregulating Angiotensin Type 1 Receptor

“…Ang II binding to AT1R is key to downstream signaling that activates intracellular second messengers and gene regulation ( Hunyady and Catt, 2006 ). Given the lack of specificity of AT1R antibodies, we used radiolabeled Ang II which is more robust and sensitive to the receptor expression ( Herrera et al, 2013 ; Nistala et al, 2013 ; Ogola et al, 2018 ). Ang II can bind to both AT1R and AT2R, and our previous studies consistently demonstrated that 2ME2 specifically downregulates AT1R and not AT2R without impacting the affinity of Ang II to the receptors ( Koganti et al, 2014 ; Ogola et al, 2018 ).…”

Angiotensin II type 1 receptor-associated protein deficiency attenuates sirtuin1 expression in an immortalised human renal proximal tubule cell line

“…Furthermore, the reactivity of NHE3 dependent on Ang II was also confirmed. Although RPTEC/TERT1 cells are an immortalised RPTEC cell line prepared using only TERT1 and are commercially available, this cell line has not been characterised regarding AT1R signalling 54,55 . Hence, our ciRPTEC will provide an alternative resource for immortalised RPTEC and could be used to analyse other proximal tubule-related proteins in addition to ATRAP.…”