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Endothelin Systems on Progressive Renal Injury To the Editor: We read the article by Cao et al 1 published recently inHypertension and have major concerns with respect to the results and interpretation of the autoradiography section. We especially refer to the kidney autoradiography study and mention for the sake of clarity only one endothelin (ET) receptor antagonist, namely, bosentan. Nevertheless, the same argument is valid for the second ET receptor antagonist used, BMS193884. In the Methods section, only a few details with respect to the autoradiography technique are provided. Even after checking the cited Reference 21, 2 some questions remain concerning the methodology. Conditions used for incubation are unclear. What molar concentration is 0.3mCi? (Incubation is normally performed at the approximate Kd of the radioligand, which for I-endothelin-1 is approximately 100 pmol). One-hour incubation in buffer containing I-endothelin-1 may be too short (most groups use a 2-hour incubation period, in which maximal binding has been achieved). Presumably, Figure 6A illustrates total radioligand binding. Nonspecific binding (in the presence of 10 25 mol/L ET-1) should also be presented. I-endothelin-1 binding in Figure 6A is somewhat regionalized, and this becomes more evident in the presence of ET antagonists. How accurate is densitometric analysis of such patchy binding? Major criticisms arise regarding the interpretation of the data. The authors are able to confirm (previously published) renal Iendothelin binding in untreated rats. A new finding is that this binding is dramatically reduced when the rats are pretreated with a single administration of bosentan. The authors’ explanation for this reduced binding (page 566) is that ET receptors are occupied as the result of the in vivo administration of bosentan, and this occupation prevents subsequent in vitro binding when sections are incubated with I-endothelin-1. We doubt that receptor occupancy by bosentan can be shown with the use of this approach. In the Methods section, the protocol used for autoradiography is based on that cited in Reference 21. In this study, all sections were preincubated in HEPES/NaCl/CaCl 2 buffer. This is usually performed to remove or reduce the endogenous ligand before the addition of radioactive ligand. When the sections are subsequently incubated withI-endothelin-1, a new steady state will be established and the radioligand may be expected to displace any remaining bound ligand, be it a receptor antagonist (such as bosentan) or ET itself. Only if one assumes such a theoretical series of events, Figure 6A (binding of radioactive ET-1 in untreated rats) can be explained: In vivo, a proportion of renal ET receptors was occupied by endogenous ET. In the preincubation step, this endogenous ET was reduced, and any remaining binding was displaced during the incubation step byI-endothelin-1, resulting in the occupation of a large proportion of ET receptors by I-endothelin-1. However, this is unlikely to occur in the bosentan experiment. The authors conclude that renal ET receptors remain occupied after in vivo administration of bosentan. This can only be explained if a very “tight” reversible, or even irreversible, binding of bosentan to receptors has occurred. Published data show that bosentan competitively binds to ET A and ETB receptors withKi values of 4.7 and 95 nmol/L, respectively. 3,4 This binding is up to 1000 times weaker than that of the endogenous ligand, ET-1 (120 and 110 pmol for ETA and ETB, respectively5). Therefore, displacement of bosentan, either by preincubation or by incubating with I-endothelin-1, should be possible. In summary, the presentation and interpretation of the autoradiographic data are inconclusive. The autoradiographic approach described in this article will only allow receptor occupancy by in vivo drug treatment to be studied if ligand binding is irreversible. Because this is not true for bosentan, the interpretation of the data is questionable. An alternative explanation for the results is that the reducedI-endothelin-1 binding that the authors describe is due to an altered receptor number and not to receptor occupation. At the time course studied, this explanation is unlikely but could be addressed by experiments of ET receptor gene expression.

Blockade of the Renin-Angiotensin and Endothelin Systems on Progressive Renal Injury