Klinische Wochenschrift

Klin Wochenschr (1988) 66:303 -307

© Springer-Verlag 1988

Binding Sites of Atrial Natriuretic Peptide in Human Renal TissueQuantification by In Vitro Receptor Autoradiography A. Brucksch I, H.J. Gr6ne 3, j. Talartschik 2, and E. Fuchs 1 1 Deutsches Primatenzentrum, G6ttingen 2 Abteilung fiir Nephrologie und 3 Abteilung fiir Pathologie, Universit~itsklinik G6ttingen

Summary. Specific binding sites for atrial natriuretic peptide (99-126) in different areas of normal human renal tissue were quantified by in vitro autoradiography. Our data represent the first characterization of A N P binding sites in different structures of the human kidney. Characterization of ANP binding revealed by Scatchard plot analysis a single class of high affinity binding sites in the glomeruli (K~ 0.53 + 0.I 1 nM; BMax74.4 _+17.86 fmol/mg protein), the vasculature (Ke 0.18+_0.014 nM; BMax 91.6--+25.02 fmol/mg protein), and the medulla (Kd 0.34_+0.13 nM; BMax 106.0--+30.61 fmol/mg protein). These sites may play a key role in the actions of the cardiac hormone in human kidney and in the ameliorating effects of A N P in the recovery from acute renal failure. Key words: A N P - H u m a n - Kidney - Receptor autoradiography

Some of the main target sites of atrial natriuretic peptide (ANP) are several renal structures. In experimental animals, A N P has been shown to cause a strong diuresis, natriuresis, and a direct vasoretaxation in the kidney [2]. Furthermore, it was demonstrated that A N P can counteract early norepinephrine-induced actue renal failure [13]. These effects of A N P are mediated through its binding to specific cell-surface receptors in different parts of the kidney. Clinical trials involving human subjects revealed a strong natriuresis and diuresis following intravenous injections of A N P [I, 11]. In contrast to the situation with experimental animals, the demonstration and characterization of putative Abbreviations: ANP=Atrial natriuretic peptide; cGMP= Cyclic guanosine monophosphate

ANP receptors in human kidneys is relatively incomplete [4, 9]. For these reasons, the discussion of the renal mechanisms and sites of action of A N P in human kidney is still open to controversy. We present the localization as well as the quantification of ANP binding sites in different parts of normal human kidneys by quantitative in vitro autoradiography. Materials and Methods Tissue from five human kidneys was investigated. Four kidney samples were tumor free parts of kidneys excised due to renal cell carcinoma in mate patients ranging from 47 to 57 years of age. One kidney from an 80-year-old female patient was obtained during autopsy 8 h post mortem. Light microscopy showed normal glomerular and tubular structures except for small focal nephrosclerotic changes in some kidneys. The tissues were frozen in isopentane, then cooled in liquid nitrogen, and stored at - 7 0 ° C. Frozen 16-~un sections were cut at - 1 8 ° C on a cryostat, thaw mounted onto gelatine-coated glass slides, and placed under vacuum at 4 ° C overnight. Binding sites tbr A N P were labeled in vitro by incubation with (3 - [ 125I]_iodotyrosil 2S)_rat A N P (specific activity 2000 Ci/mmol, AmershamBuchler, Braunschweig, FRG). In a pilot study, consecutive sections of human renal tissue were incubated either with 125I-ratA N P or t25I-human-ANP, respectively. Since no differences in binding characteristics of 125I-rat and human 125I-ANP were found, 125I-rat-ANP was used. Incubation was carried out according to Quirion et al. (1984). In short, consecutive tissue sections were preincubated at 20°C for 15 min in 50 m M Tris-HC1 buffer (pH 7.4), and then incubated for 60 min at room temperature in 50 m M

304

A. Brucksch et al. : Quantification of ANP Binding Sites in Human Kidney

Fig. 1. a Photomicrograph of the distributions of 12SI-ANP binding sites in a human kidney. The section was incubated with 150 pM ~25I-ANPand exposed for 4 days on Ultrofilm. Bar represents 5 mm. b Adjacent section incubated with 150 pM IzSI-ANP and 150 nM ANP-(99-126) to illustrate nonspecific binding. G: Glomerulus; M: Medulla: RPV: Renal preglomerular vessel; V: Renal vessel Tris-HC1 buffer p H 7.4, containing 100 m M NaC1, 5 m M MgClz, 0.5% BSA, 40 gg/ml bacitracin, 4 gg/ml leupeptin, 2 gg/mt chymostatin, 0.5 gg/ml P M S F , and lzsI-ANP in concentrations ranging from 10 to 650 pM. Nonspecific binding was determined on alternate sections in the presence of a 1000-fold excess of unlabeled rat-ANP (99-126; Peninsula Lab., Belmont, Calif., USA). Following incubation, the slides were washed three times, 2 min each, in Tris-HC1 buffer at 4 ° C and dried under a stream of cold air. To further test whether specific 1zSI_AN P binding occurred, adjacent kidney slices were incubated with 10 -~10 M labeled ligand and in a dose-dependent manner ranging from 10 -12 M to 10 -6 M with unlabeled A N P (99-126), or with the biologically inactive fragment A N P (111-126), or with A N P unrelated compounds such as angiotensin II, propranolol, and substance P. Quantification of ANP-binding sites was performed by autoradiography with 3H-Ultrofilm (LKB, Miinchen, F R G ) , computerized densito-

metry (ASBA, Leitz, K61n, FRG), and comparison w i t h 125I standards according to Israel et al. [5].

Binding data were calculated and Scatchard plots were produced by linear regression. All data are presented as the mean + S.D. For statistical analysis, data were subjected to the two-tailed MannWhitney U-test. In order to define the anatomical relationships between A N P binding sites and renal structures more exactly, we used the coverslip technique [7]. Following incubation of renal sections with radioactive 150 p M ANP, the dry sections were covered with K o d a k N T B 3 emulsion. Slides were stored at 4 ° C in the dark for 4 to 8 days. The emulsions were developed using standard methods and the tissues were stained with methyl green. Results

The autoradiographic localization of lzsI-ANP binding sites in the human kidney is shown in Fig. 1. In the cortex, glomeruli and vessels can

A. Brucksch et al.: Quantification of ANP Binding Sites in Human Kidney

305

Fig. 2a, b. Localization of 125I-ANP binding sites in a glomerulus (a, bar represents 50 gm) and a renal artery (b, bar represents 100 gm). The sections were incubated with 150 pM 125I-ANP and coated with Kodak NTB 3 emulsion. Following development, they were stained with methyl green

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Fig. 3. a Saturation curves of specific 125I-ANP binding to glomerula (v), vessels (o), and medulla (+) of human kidney. Tissue sections were incubated for 60 min with xzSI-ANP at concentrations from 10 to 650 pM. b Scatchard analysis of specific lzSI-ANP binding to medulla, glomeruli, and vessels of human kidney. These data represent a single representative experiment which was repeated for five kidneys (see Table 1); r: correlation coefficient

clearly be distinguished from the background (Fig. la) and binding is displaceable with an excess of unlabeled A N P (Fig. lb). In the outer medulla, gray structures appear in a punctuate pattern; in the inner medulla they display a diffuse picture (Fig. la). Binding in the medulla is not totally displaceable with a 1000-fold excess of unlabeled ANP (Fig. lb). The binding patterns were the same in all five kidneys. The coverslip technique enabled us to localize binding sites with high precision. Figure 2 demonstrates the distribution o f silver grains over histologically stained sections. Grains are accumulated over a glomerulus (Figure 2a) and a renal artery

(Fig. 2b). In the glomerulus, the grains are concentrated over the capillary network; in the artery, they are mainly found over the muscle layer. No accumulation of silver grains was observed when an excess of the unlabeled peptide had been added (data are not shown), Saturation curves and Scatchard analyses were performed using consecutive sections from individual kidneys. The binding kinetics of 125I-ANP in glomeruli, renal vessels, and renal medulla are shown in Fig, 3. The results obtained for saturation curves revealed that binding was saturable in all three structures (Fig. 3 a). The Scatchard plots are shown in Fig. 3 b. F r o m these plots we calcu-

306

A. Brucksch et al. : Quantification of ANP Binding Sites in Human Kidney

Table i. 125I-ANP (99-126) binding sites in human kidney.

Values represent mean_+ SD of five kidneys. Complete Scatchard plots were determined using consecutive sections from each organ (see Materials and methods) Area

Glomeruli Renal vessels Medulla

Ke (nM)

B~,,x (fmol/mg protein)

0.53 + 0.11 0.18+_0.014 0.34 +_0.13

74.4-t- 17.86 9t.6+_25.02 106.0 +_30.61

lated the maximal binding capacities (BMax) and the affinity constants (Kd). As can be seen in Table 1, binding sites in the renal vessels have the highest affinity for ANP, followed by the medulla and the glomeruli. These differences in the Kd values are statistically significant for vessels versus glomeruli (P<0.02) and vessels versus medulla (P<_0.02). In comparison with the affinities, the numbers of binding sites (BMax) are not significantly different. Specificity of 125I-ANP binding was tested by examining whether the binding could be displaced with unlabeled ANP, the truncated biologically inactive ANP (111-126), angiotensin II, or propranolol. In the presence of 10 - l ° M labeled ligand, binding of unlabeled ligand competes in a dosedependent manner in the glomeruli, the medulla, and the vessels. Less than 2% of the binding in the glomeruli is not displaceable. Unspecific binding remains _<10% in the vessels, and in the medulla in the range of 40% of total binding (Fig. 4a). No competition occurs in the glomeruli with 10-6 M angiotensin II and propranolol. Only a minor displacement can be observed with substance P and biologically inactive ANP (111-126;

Fig. 4b). Similar results are obtained in the medulla and in the renal vessels (data not shown). Discussion

The present study demonstrates, for the first time, the localization and the characterization of ANP binding sites in different parts of the human kidney by quantitative in vitro autoradiography. Binding sites are concentrated in the glomeruli, the renal vessels, and the renal medulla. Specificity of the binding sites for ANP is shown in competition experiments. From investigations of other authors, ANP is known to block the responses to angiotensin II and norepinephrine [6]. Therefore, one could assume that ANP interferes with binding of these hormones or that the binding sites might even be the same. However, our data showed no evidence for such an identity of binding sites, but allow the conclusion that ANP interacts with its own binding sites independently from other substances. In glomeruli, the affinity of ANP binding sites is consistent with results from a study by Ishikawa et al. [4]. Using membrane fractions of human renal cortex they found a Kd of about 0.4 nM. Our study indicates, however, that within the renal cortex the vessels have a significantly higher affinity for ANP than the glomeruli. As is known from animal studies, one of the prominent effects of ANP is its action on the vascular wall [15]. Therefore, one may speculate that in the human kidney, ANP causes effects in part by alterating renal hemodynamics. Furthermore, this could be one explanation of the counteracting influences of ANP following norepinephrine-induced acute renal failure [13].

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A. Brucksch et al. : Quantification of ANP Binding Sites in Human Kidney

In the renal medullary collecting duct system, A N P supposedly inhibits sodium reabsorption [14]. Due to the diffuse binding in the human medulla, our results do not allow an exact localization of A N P binding in relation to the collecting ducts. Further experiments have to be performed to discriminate whether binding sites in this part of the kidney are located on the collecting duct system or on the vasa recta system. Recent biochemical studies proposed the existence of two functional subtypes of A N P receptors [8]. One type is coupled to c G M P formation, the other " u n c o u p l e d " form of the receptor has obviously a higher affinity [15]. Because we used an equilibrium binding analysis in our study, we were unable to differentiate between these two forms and detected only one single receptor. In clinical studies it has been demonstrated that under conditions o f volume overload, such as chronic and renal failure, plasma concentrations of ANP, which is known to cause natriuresis and diuresis, are elevated [3]. One explanation for this finding might be that following prolongated elevation of plasma A N P the receptors in target organs such as the kidney may undergo an alteration of their sensitivity, their numbers, or both. A downregulation of A N P receptors was demonstrated recently in cultured vascular smooth muscle cells [12]. Therefore, our observations merit further studies on the role and functional involvement of renal A N P receptors in pathophysiological conditions such as prevention and/or amelioration of ischemic renal injury.

Acknowledgement: The authors wish to thank Mrs. G. Schmidt for her excellent secretarial assistance

References 1. Biollaz J, Bidiville J, Di6zi J, Waeber B, Nussberger J, Brunner-Ferber F, Gomez HJ, Brtmner HR (1987) Site of the action of a synthetic atrial natriuretic peptide evaluated in humans. Kidney Int 32:537-546 2. Currie MG, Geller MD, Cole BR, Boylan JG, YuSheng W, Holmberg SW, Needleman P (1983) Bioactive cardiac substances: potent vasorelaxant activity in mammalian atria. Science 221 : 71-73 3. Eisenhauer T, Talartschik J, Scheler F (1986) Detection of fluid overload by plasma concentration of human atrial natriuretic peptide (h-ANP) in patients with renal failure. Klin Wochenschr [Suppl VI] 64:68---72

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4. Ishikawa Y, Umemura S, Yasuda G, Uchino K, Shindou T, Minamizawa K, Toya Y, Kaneko Y (1987) Identification of an atrial natriuretic peptide specific receptor in human kidney. Biochem Biophys Res Coml:aun 147:135-139 5. Israel A, Plunkett CM, Saavedra JM (1985) Quantitative autoradiographic characterization of receptors for angiotensin II and other neuropeptides from individual brain nuclei and peripheral tissues from single rats. Cell Mol Neurobiol 5: 211-222 6. Kleinert HD, Maak T, Atlas SA, Januszewicz A, Sealey JE, Laragh JE (1984) Atrial natriuretic factor inhibits angiotensin-, norepinephrine-, and potassium-induced vascular contractility. Hypertension (Suppl I) 6:I-143 1-147 7. Kuhar MJ (1985) Receptor localization with the microscope In: Yamamura ItI, Enna SI, Kuhar MJ (eds) Neurotransmitter receptor binding. Raven Press, New York, pp 25~270 8. Leitman DC, Andresen JW, Kuno T, Kamisaki Y, Chang JK, Murad F (t986) Identification of multiple binding sites for atrial natriuretic factor by affinity cross-linking in cultured endothelial cells. J Biot Chem 261 : 11650-I 1655 9. Mantyh CR, Kruger L, Brecha NC, Mantyh PW (1986) Localization of specific binding sites for atrial natriuretic factor in peripheral tissues of the guinea pig, rat, and human. Hypertension 8:712-721 10. Quirion R, Dalpe M, de L6an A, Gutowska J, Cantin M, Genest J (1984) Atrial natriuretic factor (ANF) binding sites in brain and related structures. Peptides 5:1167-1172 11. Richards AM, Nicholls MG, Ikram H, Webster MW, Yandle TG, Espiner EA (1985) Renal, haemodynamic, and hormonal effects of human alpha atrial natriuretic peptide in healthy volunteers. Lancet I: 545-549 12. Roubert P, Lonchampt MO, Charbier PE, Plas P, Goulin J, Braquet P (1987) Down-regulation of atrial natriuretic factor receptors and correlation with cGMP stimulation in rat cultured vascular smooth muscle cells. Biochem Biophys Res Commun 148: 61-67 13. Schafferhans K, Heidbreder E, Grimm D, Heidland A (1986) Norepinephrine-induced acute renal failure: Beneficial effects of atrial natriuretic factor. Nephron 44: 240-244 14. Sonnenberg H, Honrath U, Chong CK, Wilson DR (1986) Atrial natriuretic factor inhibits sodium transport in medullary collecting duct. Am J Physiol 250 : F963 F966 15. Vlasuk GP, Babilon RW, Nutt RF, Ciccarone TM, Winquist RJ (1987) The actions of atrial natriuretic factor on the vascular wall. Can J Physiot PharmacoI 65:1684-1689

Received: October 8, 1987 Returned for revision: November 24, t987 Accepted: January 5, 1988

Dr. E. Fuchs Deutsches Primatenzentrum Kellnerweg 4 D-3400 G6ttingen