In vitro autoradiographic localization of [125I]-angiotensin II binding sites in the rat and dog kidney

Peptide.~. Vol. 5, pp. I(H3-I048. 1984. ' Ankho International Inc. Printed in the U.S.A.

0196-9781/84 $3.00 + .00

In Vitro Autoradiographic Localization

of [azsI]-Angiotensin II Binding Sites in the Rat and Dog Kidney D. R. GEHLERT, R. C. S P E T H 1 A N D J. K. W A M S L E Y " Departments o f Pharmacology and Psychiato', University o f Utah Medical Center, Salt Lake City, U T 84132 R e c e i v e d 7 M a y 1984 GEHLERT, D. R., R. C. SPETH AND J. K. WAMSLEY. In Vitro autoradiographic localization of [v-':'l]-angiotensin i1 binding sites in the rat and dog kidney, PEPTIDES 5(6) 1043--1048. 1984.--Light microscopic autoradiographic techniques have been utilized to demonstrate specific regions of the rat and dog kidney where angiotensin II receptors exist. Slide mounted tissue sections were labeled with [~ZSl]-angiotensin11 using conditions which provided for highly specific binding. These angiotensin II binding sites were localized to several distinct renal structures. In the renal cortex, angiotensin II binding sites were found concentrated in all parts of the glomeruli including the vascular components, the macula densa and the juxtaglomerular apparatus. Angiotensin II binding in the medulla was more diffusely associated with the vasa recta, and to a lesser extent, the thick ascending segment of the loop of Henle. Binding sites specific for angiotensin 1I were also found in the smooth muscle laminae of the ureter. Scatchard analysis of the binding kinetics allowed the demonstration of two subpopulations of binding sites which differ slightly in their affinities for [~251]-angiotensinI1. These subpopulations appear to be associated with distinct components of the renal structure. Receptor autoradiography Angiotensin I1 receptors Hypertension Renal function Glomerular angiotensin II receptors Humoral angiotensin I1 Renal hypertension Angiotensin I1 receptors in kidney Rat kidney receptors

rat and dog kidney utilizing in vitro receptor autoradiography [23, 36, 37].

T H E involvement of angiotensin II in hypertension is well documented [19]. Angiotensin II has a variety of physiologic roles, some of the most prominent being in the kidney. It produces alterations in glomerular filtration rate (GFR) by affecting arteriolar resistance [9, 13, 25] and may contract the mesangial cell layer to decrease glomerular capillary surface area [15.30]. Angiotensin II also appears to be an intermediate in the actions of dibutyryl cAMP, parathyroid hormone. prostaglandin I~ and prostaglandin E~ resulting in a decrease of plasma flow rate and an increase in total renal arteriolar resistance [3]. Using a variety of techniques, an increase in proximal tubular reabsorption was found to be directly mediated by angiotensin II [14, 16, 17, 24]. There is some suggestion that angiotensin II can alter reabsorption in the late proximal tubule, early distal tubule, and the loop of Henle as well [24.25]. Angiotensin II also mediates a feedback inhibition on renin release by the juxtaglomerular apparatus [ 10.28]. These studies implicate a potential role for kidney receptor populations in angiotensin induced hypertension. With these possibilites in mind. the present study was undertaken to localize the potential sites of action of angiotensin II in the

METHOD Male, Sprague-Dawley rats (200-250 g) were sacrificed by intracardial perfusion with ice cold sodium phosphate buffered saline (0.01% formalin) at p H 7.4. Kidneys were removed and frozen onto brass cryostat chucks coated with OCT compound (Lab-Tek Products; Naperville, IL). Sections (10-16 microns) were cut on a Harris cryostat microtome (Harris; North Billerica, MA) at -15°C and thawmounted onto cold chrome/alum subbed slides. The kidneys from two mongrel dogs were treated in a similar manner. Serial sections were preincubated in a media containing 0.4% bovine serum albumin, 10raM MgC12, 150 mM NaC1, 5 mM E G T A , 5 mM dithiothreitol, 30 mM N~HPO~ at pH 7.1 for 30 minutes. After preincubation, the slides were incubated in the same media containing 0.5 nM [12sI]-Ileangiotensin II ( S A = 1785 Ci/mmole) for 60 minutes. Sections representing nonspecific binding were generated by incubating sections in the presence of 3 micromolar unlabeled Ile sangiotensin II or ValS-angiotensin II. Incubations were ter-

'Section of Cardiovascular Neurobiology. Department of Cardiovascular Research, The Cleveland Clinic Foundation, Cleveland, OH 44106, :Requests for reprints should be addressed to James K. Wamsley. Ph.D.. Department of Psychiatry, The University of Utah School of Medicine. 50 North Medical Drive. Salt Lake City. UT 84132.

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FIG. 1. Autorediographic images on LKB Ultrofilm after apposition to kidney sections labeled with [mI]-angiotensin II. A. A photomicrograph of the autoradiographic grains on Ultrof'dmapposed over a kidney section incubated in the presence of 0.2 nM [~l]-angiotensin II. Note the high density of autoradiographic grains associated with the giomerulus (81) and vasa recta (vr). B. The tissue section used to generate the autoradiogram depicted in this photomicrograph was incubated in the presence of 1.3 nM [r~'~I]-angiotensinII. Note the increased binding to areas outside the vasa recta in the medulla (M). C. The autoradiogram depicted in this photomicrograph was produced from a section incubated with 0.2 nM [~'I]-angiotensin II in the additional presence of 3 micromolar ValS-angiotensin II. The diffuse autoradiographic grains seen here represent nonspecific binding. Bar=500 microns.

minated by a dip and two 5-minute rinses in fresh media (without added radioactivity) at 4°C. The slides were then dipped twice in distilled water, placed on ice cold metal pans and dried with a stream of cool, dry air. Saturation studies were performed using sections of rat kidney by varying the concentration of [nH]-angiotensin II in the incubation media from 0.1 nM to 1.3 nM. After overnight desiccation, the slides were affixed to photographic mounting board and apposed to LKB Uitrofdm (LKB Instruments; Rockville, MD) in X-ray cassettes for 2-4 days, after which the f'dm was removed and developed. To facilitate quantitation in the saturation experiments, [1~I] brain paste standards were prepared using di-iodo-angiotensin II in a manner similar to that for tritium brain paste standards as previously described [35]. Exposure of these standards were included on each sheet of film. Some slides were also positioned against emulsion coated coverslips [37]. This method entailed affixing emulsion (NTB-3, Eastman Kodak; Rochester, NY) coated coversfips over the sfide mounted tissue sections. After a six-day exposure, the coverslips were bent back, the latent images were developed and the tissue subsequently stained. The coverslips were then permanently affixed to the slide, positioning the autoradiographic grains directly over the source of radioactivity in the tissue. Thus, a high resolution and an accurate localization of the autoradiographic grain distribution could be obtained. The latent images and stained tissue sections were photographed on a Leitz Orthoplan microscope equipped with an Orthomat Camera System (Leitz; West Germany). Grain density readings on the LKB Ultrofilm were made with a computer assisted microphotometry system (DADS Model

560) attached to the Leitz microscope. In the saturation experiments, these readings were converted to femtomoles ligand bound/mg tissue by comparing the grain densities over the tissue areas to those produced by [ ~ I ] brain paste standards [35]. [n'~I]-Ile'~-angiotensin II was synthesized (R.C.S.) by a previously described method [29]. Purity was confirmed by thin layer chromatography, high performance liquid chromatography and receptor binding techniques. RESULTS Incubation conditions, similar to those described here, have been used previously to define areas of high specific binding of [nsI]-angiotensin II in the rat brainstem [ 12]. Preliminary experiments indicated that the binding of [~2~I]angiotensin II to rat kidney took place in a similar fashion. The use of these conditions produced highly specific binding, represented by autoradiographic grains on LKB Ultrofilm, in several kidney areas (Fig. IA and 1B). The addition of either VaD-angiotensin II or Ile~-angiotensin II to the preincubation and incubation medias produced an image that had few autoradiographic grains in these areas (Fig. IC). Highly specific grain densities, representing p"'~I]-angiotensin II binding to angiotensin II receptors, were seen in areas on LKB Ultrofilm that correspond to the glomeruli, cortex, medulla and the smooth muscle of the ureter (Figs. 1 and 2, and Table l). The use of the coverslip technique provided for the localization of angiotensin II binding in distinct parts of the glomerulus including the vasculature, macula densa and juxtaglomerular apparatus (Fig. 3). These sites could be further localized to the basement membrane and mesangial cells of

L O C A L I Z A T I O N O F A N G I O T E N S I N II RECEPTORS

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F I G . 2. [ l ~ I ] - a n g i o t e n s i n II binding in the ureter. A. A tissue section stained with cresyl violet is seen in this photomicrograph. The arrow denotes the location of the rat ureter (V). B. A portion of Ultrofiim apposed over the tissue section seen in A is seen in this photomicrograph. Note the high density of autoradiographic grains corresponding to the ureter. Bar=500 microns. C. The labeled tissue section used to produce this autoradiogram was sectioned at a fllore medial aspect than those seen in A and B. Again, the high grain density associated with the ureter can be appreciated. Bar=500 microns.

the glomeruli. In the renal medulla, angiotensin II binding appeared to be associated with the vasa recta and, to a lesser extent, the thick ascending limb of the loop of Henle. Saturation studies performed on film gave evidence for two receptor affinities (Fig. 4); a high affmity receptor which was associated with the vasa recta, and a lower a~inity receptor seen in areas of the medulla outside the vasa recta. Receptor populations in the ureter had a dissociation constant (KD) similar to that observed in the vasa recta, while the population in the glomerulus resembled the KD measured in the medulla outside the vasa recta. Slides incubated in the presence of 3 micromolar IleS-angiotensin II or 3 micromolar ValS-angiotensin II had low amounts of autoradiographic grains in these areas. Binding to dog kidneys was localized to similar regions (Table 1), however the binding was more diffuse in the renal medulla and could not be localized specifically to any tubular or vascular populations. There was also no specific binding corresponding to the ureters in the sections of dog kidney we examined.

DISCUSSION

In vitro receptor autoradiographic techniques have been appfied to localize angiotensin II receptors in the rat and dog

TABLE 1 DENSITOMETERREADINGSOF GRAINDENSITIESON LKB ULTROFILMEXPRESSED AS PERCENTEXTINCTION Area

Total

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% Specific

Rat* Glomerulus Cortex Vasa Recta Medulla Ureter Dogt

62.8 24.7 60.6 28.2 66.7

± ± ± ± ±

2.9 1.1 2.6 1.0 1.5

16.5 ± 18.2 ± 6.2 ± 8.6 ± 6.8 ±

0.5 0.7 1.1 0.8 1.2

73.7 23.5 89.9 69.5. 89.8

Glomerulus Cortex Medulla Ureter

74.0 32.2 34.2 33.7

± ± ± ±

1.8 2.3 1.6 2.7

20.8 ± 13.8 ± 10.3 ± 29.3 ±

0.9 2.1 0.8 2.7

71.9 57.1 69.9 N.S.

*N--6. iN--2. Readings were taken from a 250 square micron window positioned over the area on fdm corresponding to the appropriate kidney area. Readings from the cortex and medulla refer to areas outside the grain densities associated with the glomeruhis and vasa recta, respectively, p<0.05.

1046

GEHLERT, SPETH AND WAMSLEY

Fig. 3. Localization of angiotensin II receptors in the rat renal cortex using the photographic emulsion-coated coverslip technique. A. After development of the latent autoradiographic image on the coverslip, the tissue section seen in this photomicrograph was stained with pyronine Y. The location of the glomerulus (gl) corresponds to the arrow. The autoradiographic grains associated with the glomerulus can be seen directly as dark grains. B. The incident light used to take this photomicrograph makes the autoradiogram appear as white grains against a dark background. Again note the high grain density associated with the glomeruins. Bar =500 microns.

kidney. Discrete grain densities, representing specific binding o f ['"sI]-angiotensin II, have been detected in areas corresponding to the renal cortex, glomerular structures, renal medulla and the ureter. The use o f coverslip techniques allowed further localization o f this highly specific binding to the giomerular basement membrane, giomerular mesangiai cells, smooth muscle lamina o f the ureter, and the vasa recta in the medulla. In vitro receptor autoradiographic techniques are particularly advantageous for the localization o f receptors for peptide neurotransmitters and hormones. The presence o f peptidase inhibitors, such as dithiothreitol, E G T A and bovine albumin provide metabofic stability so the bound peptide remains intact. This assures that the specific binding represents actual [;25I]-angiotensin II binding rather than labeled peptide fragments which can occur, due to metabolism, when using in vivo techniques. Nonspecific binding of labeled peptide fragments is also reduced, allowing more precise measurement of the specific angiotensin II recognition sites in the tissues. The receptor labefing is thus performed under optimal conditions to produce a high degree of specific binding (Table 1). The renin-angiotensin system has been implicated in several types o f hypertension, with the kidney being a source of angiotensin as well as one o f the major sites of angiotensin's action [8,18]. Physiologically, angiotensin II exerts a variety of effects on the kidney. It is a potent systemic vasoconstrictor and hypertensive agent, increasing both systemic "and renal vascular resistance [9, 27, 38]. However, the specific effects o f angiotensin II on renal hemodynamics is presently ambiguous. It appears to cause an alteration in the muscle tone o f the afferent or efferent arteriole which can produce an increase or decrease in glomerular filtration rate [2, 10, 11, 13, 16, 21]. The presence o f angiotensin II receptors in the renal vasculature provides additional evidence for the ability of angiotensin II to alter renal vascular tone. Saturation studies indicate two sites o f similar Braax, but different affinities exist in the kidney. A high affinity receptor population was found in the vasa recta and ureter. A slightly lower affinity receptor population was localized to the giomerulus and medulla outside the vasa recta, presumably associated with tubular populations. The high

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BOUND FIG. 4. Scatchard analysis of [r-':'l]-angiotensin II binding to the rat kidney. Serial rat kidney sections were incubated in the presence of 0.1-1.3 nM concentrations of iodinated angiotensin II and apposed to Ultrofilm for 6 days. [~=sI] Brain paste standards, containing known concentrations of [;='~I] were placed on the film with the labeled tissue sections. Densitometric measurements (using computer assisted microdensitometric techniques) were convened to fmoles [~nI]-angiotensin II bound/rag tissue by comparing the grain densities corresponding to the appropriate tissue areas to the grain densities produced by the standards. Scatchard analysis of this data gave evidence for two binding affmides. Similar data (with each point varying less than 10%) were obtained from each of four subsequent experiments encompassing quadruplicate serial sections of kidney from six different animals. A high affinity receptor population (KD=0.23 riM, Bmax= 14.5-16.0 fmoles/mg tissue) and a low affinity population (Ko=0.77-0.85 nM, Bm,,x=16.5-17.5 fmoles/mg tissue) which were localized to distinct kidney structures (see inset). These data were originally computed and drawn using a HewletbPackard 9845C minicomputer interfaced with an X-Y plotter. The lines represent linear regression analysis of the points and each shows a correlation coefficient in excess of 0.97.

L O C A L I Z A T I O N OF A N G I O T E N S I N I1 RECEPTORS affinity binding appears to be associated with the smooth muscle in the vasculature and ureter while the lower affinity binding could be associated with other types of tissue. These results indicate either two distinct receptor populations or two different affinity states of the same receptor exist for angiotensin I1 in subcomponents of the renal structure. Angiotensin also appears to cause a reduction in glomerular size, an effect which may be mediated by glomerular mesangial cell contraction [15,30]. Previous binding studies utilizing homogenate techniques have demonstrated specific binding of labeled angiotensin II in isolated glomeruli [4, 7, 30] and these binding sites were further localized to the basement membrane [31 ]. The use o f in vivo autoradiography after systemic administration of labeled angiotensin I1 has also resulted in the localization of binding to the glomerular mesangial cells [22]. However, in this in vivo study, no binding to arterioles or tubular elements was reported. Our use of an in vitro technique demonstrates specific binding not only in the glomerular basement membrane and mesangial cells, but in the vasa recta and ureter as well. One can conclude that these additional receptor populations may be inaccessible to circulating angiotensin II or that radiolabeled angiotensin II administered systemically is degraded either before reaching these sites or before the tissues can be fixed. Using the coverslip technique, a low density of autoradiographic grains was seen in the region of the proximal tubule and in the areas of the medulla containing the loop of Henle. The results indicate that angiotensin II receptors may be associated with these tubular elements. This finding supports physiological data on the effects of angiotensin II and its analogs which can directly increase the proximal tubular reabsorption rate [14, 16, 17]. Angiotensin II has also been implicated in alterations of reabsorption in the segment between the late proximal and early distal tubule [24-26]. The localization of autoradiographic grains in areas corresponding to the ascending loop of Henle implicate this segment as the site of action. Since specific angiotensin II binding has

1047 been demonstrated in isolated rat renal brush border membranes I5], our results indicate that these receptors are probably on the luminal side of the proximal tubule and are presumably inaccessible to circulating hormone. Immunoreactive angiotensin I! has been detected in the kidney in several studies. Histochemical techniques have localized angiotensin-like immunoreactivity in the juxtaglomerular cells, afferent arteriole and mesangial cells [20, 32-34]. These structures could release angiotensin 11 in order to activate the angiotensin II receptors we identified in each corresponding region. However, angiotensin II receptors have also been localized in the medulla and ureters. The functional significance of the binding to the ureter is unknown. However, since smooth muscle is a major component of the ureter, and since angiotensin II receptors have been demonstrated to exist in other nonvascular smooth muscles including the bladder [1], it follows that angiotensin II may function to contract the ureter to move urine to the bladder. The binding to the tubular elements in the medulla may affect tubular reabsorption, as has been discussed above. The use o f in vitro receptor autoradiography in this study has uncovered the existence of angiotensin II receptors in areas where their presence was previously unidentified. The examination of the dissociation constants and receptor numbers as described here, would be virtually impossible using any other technique currently available. These observations, coupled with evidence for a synthetic and degradative capacity [6, 20, 34] within the kidney, gives new credence to an intra-renal role for the renin-angiotensin system.

ACKNOWLEDGEMENTS The authors wish to express their gratitude to Jane Stout and Rosalyn Fowles for their excellent secretarial assistance. This research was supported by Public Health Service grants MH-36563, HL-27568 and HL-6835,

REFERENCES

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