Kidney International, Vol. 42 (1992), PP. 1372—1380

High resolution localization of angiotensin II receptors in ratrenal medulla

JIALONG ZHUO, DAINE ALCORN, ANDREW M. ALLEN, and FREDERICK A.O. MENDELSOHN

Department of Medicine, University of Melbourne, Austin Hospital, Heidelberg, and Department of Anatomy, University of Melbourne,Parkville, Victoria, Australia

High resolution localization of angiotensin II receptors in rat renalmedulla. The cellular localization of angiotensin II (Ang II) receptors inthe inner stripe of the outer medulla of the rat kidney was investigatedby using high resolution light and electron microscopic autoradiogra-phy. Fresh tissue blocks from the inner stripe of the outer medulla wereincubated with '251-[Sar', lie8] Ang II and prepared for microscopicautoradiography. At the light microscopic level, '251-[Sar', lie8] Ang IIwas found to penetrate into the tissue and to bind specifically to sitesoutlining renal tubules and vasa recta bundles. Electron microscopicautoradiography revealed that silver grains were detected over intersti-tial cells located between the tubules and components of the vasa rectabundles, but no silver grains were detected overlying the cells of thethin descending or thick ascending limbs of the loop of Henle, thecollecting ducts, the vasa recta, or other blood vessels. These intersti-tial cells contained abundant endoplasmic reticulum, microfilaments,occasional lipid droplets and extensive cytoplasmic processes whichclosely related to the basement membranes of the vasa recta and loopsof Henle. The cells therefore closely resemble type 1 interstitial cells.Since Ang II binding sites are absent in the inner medulla, the cellslabelled by this technique must be a subset of type 1 interstitial cells,distinct from the typical lipid-laden interstitial cells most abundant inthe inner medulla. These findings demonstrate that type 1 interstitialcells are the primary sites for a high density of Ang II receptors locatedin the inner stripe of the outer medulla.

The renal medulla plays a fundamental role in maintainingbody fluid and electrolyte homeostasis through the medullarymicrocirculation and through urinary concentrating mecha-nisms [1—3]. Angiotensin II (Ang II), among several othervasoactive peptides, has been implicated in regulating theseprocesses [4—8]. In the renal medulla, Ang II stimulates pros-taglandin production [9] and phosphatidylinositol and polyphos-phoinositide turnover [101. Activation of the renin-angiotensinsystem in sodium-depleted dogs [11], or direct intrarenal infu-sion of Ang II, markedly reduces medullary and papillaryplasma flow [12], whereas pharmacological blockade of thesystem by converting enzyme inhibitors, captopril, or by Ang IIreceptor antagonist, saralasin, increases medullary blood flow[6—8, 12]. These studies suggest that Ang II may modulate thecountercurrent system and urinary concentrating process by itsactions on renal meduliary microcirculation.

Received for publication December 20, 1991and in revised form June 24, 1992Accepted for publication June 25, 1992

© 1992 by the International Society of Nephrology

Consistent with the actions of Ang II in the renal medulla, wehave previously demonstrated the presence of abundant highaffinity Ang II receptors in membrane fractions prepared fromrat renal outer medulla [13]. These receptors have been consis-tently confirmed by in vitro autoradiographic studies [13—15].One of the most striking features of these in vitro autoradio-graphs is a moderate high density of Ang II receptor bindingdiffusely throughout the inner stripe with additional longitudinalbands of very high density binding traversing this region. Thesebands correspond to the pale bands in hematoxylin-eosinstained sections, and showed a strongly-positive reaction prod-uct for the vascular endothelial marker, factor VIII, or immu-noperoxidase labelling suggesting that the distribution of Ang IIreceptors to the inner stripe is higher in association with thevasa recta bundles [15]. However, the specific cellular localiza-tion of Ang II receptors in the inner stripe of the outer medullahas not previously been determined.

Although in vitro autoradiography of frozen dehydratedkidney sections reveals a high density of Ang II receptors in theinner stripe of the outer medulla, it lacks adequate resolution tolocalize receptors to specific cells [13—15]. Previous studieshave shown Ang II receptor binding on rabbit renomedullaryinterstitial cells in tissue culture [16]. However, these cells aremost abundant towards the tip of the inner medulla [11, adistribution which does not correlate with the anatomical dis-tribution of renal Ang II receptors which are very low in theinner medulla [13—15]. The present study was, therefore, under-taken to localize Ang II receptors at the cellular level in theinner stripe of the outer medulla by using high resolution lightmicroscopic autoradiography and electron microscopic autora-diography.

Methods

In vitro autoradiographic localization of renal Ang IIreceptors in frozen tissue slices

Adult male Sprague-Dawley rats (200 g body wt) were killedby decapitation. The kidneys were removed quickly, snap-frozen in isopentane at —40°C and stored at —80°C. Sections (20sm thick) were cut on a cryostat at —18°C, dehydrated over-night under reduced pressure at 4°C, and then stored at —80°Cin sealed containers with silica gel. Autoradiographic localiza-tion of renal Ang II receptors was performed by using ourroutine procedures [13—15]. Briefly, Ang II receptor bindingsites were labelled with the antagonist analogue, '251-[Sar', lie8]

1372

Zhuo et a!: Ang II receptors in rat renal medulla 1373

F1F.1. In vitro autoradiographic localization of Ang II receptors in rat kidney. Longitudinal sections were processed and incubated with

12 I-[Sar', lie8] Ang II as previously described [15, 18]. Abbreviations are: G, glomerulus; PCT, proximal convoluted tubule; IS, inner stripe of theouter medulla; IM, inner medulla. A. Total binding. B. Non-specific binding in the presence of an excess (1 M) of unlabelled Ang II. Ang IIreceptor density is indicated by white silver grains on a dark background.

Ang II. The slide-mounted sections were preincubated for 15minutes in 10 mrvi sodium phosphate buffer (pH 7.4), followedby a one hour incubation in fresh volume of the same buffercontaining 0.2 CiIml (—90 pM) '251-[Sar', Ile8I Ang II, 0.2%bovine serum albumin and 0.3 mivi bacitracin. Nonspecificbinding was determined in parallel incubations in the presenceof 1 pM unlabelled Ang II. After incubation, the sections werewashed four times for one minute each in ice-cold buffer, driedunder a stream of cold air and exposed to Agfa Scopix CR3BX-ray film at room temperature for 48 to 72 hours. A set ofradioactivity standards were included in each cassette. Follow-ing exposure, the films were processed and the optical densitiesquantitated by a microcomputer imaging device (MCID Imagingsystem, Ontario, Canada) coupled to an IBM AT computer. Acalibration curve of optical density versus radioactivity densitywas constructed by the computer program using the standards.This enabled remapping of the autoradiographs into dpm/mm2[15]. The receptor distribution revealed by this in vitro autora-diographic method was compared with that observed by highresolution light and electron autoradiography.

High resolution light and electron microscopicautoradio graphic localization of renal Ang II receptors in

fresh tissue slices

Adult male Sprague-Dawley rats (-—-100 g body wt) werekilled by decapitation. The kidneys were removed quickly andsliced using a scalpel into 1 mm thick slices. Blocks of 1 mm X1 mm by 5 mm long were cut from the inner stripe of the outermedulla, and preincubated in Krebs buffer saturated with 95%02:5% C02, (pH 7.2) containing 124 mM NaC1, 26 mMNaHCO3, 3 mzi KCI, 1.4 mri KH2PO4, 2.4 mM CaCI2, and 4m glucose for 30 minutes at 22°C. The blocks were thenincubated in the same buffer containing 5 ma'i EGTA, 0.2%BSA, and 260 M 1251-[Sar', lIe8] Ang II at room temperaturefor one hour. Following incubation, the blocks were washed infour changes of ice-cold buffer, each for five minutes, with thetissues free floating in plastic dishes. The buffer was removed

by suction. Non-specific binding was determined in parallelincubations in the presence of 1 LM unlabelled [Sar', lIe8] AngII under similar conditions to those described above.

Upon completing the washes, the tissue blocks were fixed in2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, at roomtemperature for one hour, and then washed twice (5 mm each)with 0.1 M phosphate buffer containing 5% sucrose, postfixed in2.5% aqueous osmium tetroxide, dehydrated in graded acetone,and embedded in Araldite-Epon (Ladd, Vermont, USA). Forhigh resolution light microscopic autoradiography, the tissueblocks were sectioned (0.7 m) and were positioned on chromalum glass slides, dipped in Kodak NTB2 emulsion (EastmanKodak, Rochester, New York, USA) and exposed at 4°C for upto 60 days. The slides were developed in Kodak D19 and lightlycounterstained with 1% methylene blue. Light micrographswere taken using both bright and dark field exposure condi-tions.

For electron microscopic autoradiography, thin sections (60to 90 nm) were cut on a diamond knife and positioned oncelloidin coated glass slides. The slides were then coated inIlford K5 emulsion (Ilford, Mobberly, Cheshire, UK) andexposed at 4°C for 60 days. After exposure, the slides weredeveloped in Kodak D19 and the sections floated onto waterand picked up onto 200 mesh grids, stained with uranyl acetateand lead citrate, and examined in a Siemens 102 Elmiskopelectron microscope.

Results

Localization of renal Ang II receptors by in vitroautoradiography

The anatomical distribution of renal Ang II receptors wasidentical to those we reported previously [13—15]. As shown inFigure IA, high densities of 1251-[Sar', lie8] Ang II bindingoccur in giomeruli and in longitudinal bands traversing the innerstripe of the outer medulla, whereas a moderate density ofbinding is seen in the interglomerular region, corresponding to

1374 Zhuo et al: Ang II receptors in rat renal medulla

Fig. 2. Light photomicrographs of thickplastic sections of transverse sections of theinner stripe of the outer medulla of the ratkidney following incubation with 260 M 125j[Sar', Ile8] Ang II for one hour, Asterisksindicate tubules surrounded with silver grainsand arrows indicate vasa recta bundleslabelled with silver grains in both dark and thecorresponding bright field. Abbreviations are:V, vasa recta bundles; T, tubules. A. Darkfield illumination showing white silver graindevelopment in specific ring-like patternsaround the outer edge of the tissue block.Magnification: X 100. B. Bright fieldillumination of the same section as in (A)showing the characteristic arrangement of thetubules and vasa recta bundles within thesection. From the comparison of these twofigures, silver grains are distributed aroundthe tubules, and around the components ofthe vasa recta bundles of the outer parts ofthe tissue block where the radioactiveanaloque has penetrated. Stain: methyleneblue; magnification: x 100. C. Dark fieldillumination at higher resolution. Specificsilver grain development is confined to theouter region of the section. Magnification:x400. D. Bright field illumination of the samesection as in (C), demonstrating that the ring-like pattern of silver grain development isfound around larger tubules as well as aroundthe components of the vasa recta bundles.The labelled analogue has not penetrated tothe center of the tissue, where both vasa rectabundles and regions of tubules can beidentified. Stain: methylene blue;magnification x400.

the proximal convoluted tubules, and the interbundle area ofthe inner stripe. Binding is not detectable in the inner medulla.Non-specific binding was very low representing approximately2% to 5% of the total binding (Fig. 1B).

High resolution localization of Ang II receptor in the innerstripe of the outer medulla following in vitro labelling

'251-[Sar', 11e8] Ang II penetrated into the tissue blocks of theinner stripe of the outer medulla and was bound to sites at theedge of the strip of tissue (Fig. 2A). In particular, in transversesections the binding formed a pattern outlining the renal tubulesand the vasa recta (Fig. 2 C and D).

At the electron microscope level grains were detected overinterstitial cells located between the distal tubules (Fig. 3A),collecting ducts (Fig. 3C) and between the components of thevasa recta bundles (Fig. 3B). These interstitial cells had multi-ple cytoplasmic processes in close association with tubules(Fig. 4A), capillaries and vasa rectae (Fig. 4B). At highermagnification the interstitial cells were observed to containmitochondria, cisternae of rough endoplasmic reticulum, freeribosomes, polysomes, lysosomes and vacuoles in the cyto-plasm and sparse lipid droplets (Fig. 4 A, B, and C). Smallbundles of microfilaments were positioned at the periphery ofthese cells. There was no labelling over cells of the thick

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Fig. 3. Electron micrographs of thin sectionsof the inner stripe of the outer medulla of therat kidney incubated with 260 M of '251-[Sar',lie8] Ang II for one hour. Abbreviations are:DT, distal tubule; IC, interstitial cell; TL, thinlimb of the loop of Henle; In, intercalated cellof the collecting duct; P, principal cell of thecollecting duct; Ca, capillary. Stain: uranylacetate and lead citrate. A. Silver grainslocalized to interstitial cells and theirprocesses between distal tubules andcapillaries. Distal tubular cells, capillaries andthe interstitial space are not specificallylabelled. Magnification: x6,750. B. Silvergrains localized to interstitial cells and theirprocesses in a vasa recta bundle. Thin limbsof the loop of Henle, capillaries andinterstitial space are not specifically labelled.Magnification: x5,250. C. Silver grainslocalized to interstitial cells and theirprocesses between collecting ducts andcapillaries. The principal and intercalated cellsof the collecting duct, the capillaries, and theinterstitial space are not specifically labelled.Magnification: x 6,750.

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1376 Zhuo et al: Ang II receptors in rat renal medulla

Fig. 4. Electron micrographs of thin sectionsof the inner stripe of the outer medulla of therat kidney incubated with 260 p of '251-[Sar',lIe8] Ang Ii for one hour. Abbreviations are:DT, distal tubule cells; IC, interstitial cell; Ca,capillary; Pe, pericyte. Stain: uranyl acetate,lead citrate. A. Silver grains localized to aninterstitial cell in a tubular region. Thecytoplasm of the interstitial cell containsmitochondria, dilated granular endoplasmicreticulum, ribosomes, vacuoles and peripheralfilments. Magnification: x 15,000. B. Silvergrains localized to an interstitial cell in a vasarecta bundle. The interstitial cell containssimilar organelles to the interstitial cell in (A)and also cytoplasmic lipid inclusions (L).Magnification: x20,000. C. Silver grainslocalized to an interstitial cell. An adjacentpericyte or type 3 interstitial cell is notspecifically labelled. The pericyte surrounds acapillary and is covered in a continuousbasement membrane (arrowheads).Magnification: x 15,000.

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Zhuo et al: Ang II receptors in rat renal medulla 1377

ascending limbs, the thin descending limbs, the collectingducts, the vasa recta or other blood vessels.

As shown in Figure 4C, pericytes or the type 3 interstitialcells surrounding the vasa recta and enclosed by a continuousbasement membrane were not specifically labelled. However,macrophages or the type 2 interstitial cells were infrequentlyseen in the inner stripe of the outer medulla in our tissuepreparation.

In sections prepared from tissue incubated in the presence of1 LMunlabelled Ang II, virtually no silver grains were observedin both light and high resolution electron micrographs, indicat-ing that non-specific binding was undetectable (Fig. 5 A to C).

Discussion

Using high resolution electron microscopic autoradiography,the present study demonstrates that the interstitial cells, ratherthan renal tubular cells, vasa rectae, or other blood vessels arethe primary sites for specific Ang II binding in the inner stripeof the outer medulla. These findings suggest a role for interac-tions between Ang II and interstitial cells in modulating med-ullary processes.

Because in vitro autoradiography on frozen dehydrated tis-sues does not give adequate spatial resolution for cellularlocalization in the renal medulla, we employed light and elec-tron microscopic autoradiography on fresh tissue which hadbeen incubated in vitro with 1251-[Sar', Tie8] Ang II. Thisimproved resolution sufficiently to unequivocally localize AngII receptors at the cellular level, but did not provide as goodtissue morphology as may be obtained with optimally fixedtissue in vivo. Nevertheless, clear-cut results were obtained.By using this technique, we observed that '251-[Sar1, Ile8] AngII was bound to sites outlining the tubules and the vasa rectabundles. Interestingly, electron microscopic autoradiographydetected silver grains only over the interstitial cells between thetubules and between the components of the vasa recta bundles.No appreciable grains were found in the other components ofthe inner stripe, including the distal tubule, collecting duct, theloop of Henle, the vasa recta and other blood vessels.

Localization of Ang II receptors to the interstitial cells in theregions of both the vasa recta bundles and the tubules by highresolution electron microscopic autoradiography was unex-pected, since our previous in vitro autoradiographic studiessuggested that Ang II receptors were preferentially located inthe vasa recta bundles [15]. This apparent paradox may beexplained by the relative volumes occupied by interstitial cellsin these two regions of the inner stripe of the outer medulla. Inthe tubular region, interstitial space occupies a relatively smallvolume compared with the large distal tubules and collectingducts, whereas in the vasa recta bundles a larger volume isinterstitial space compared with the smaller diameter thin limbsand vascular elements [1, 17]. Thus, while silver grains will beobserved throughout the inner stripe of the outer medulla, therewill be a greater density of grains associated with the vasa rectabundles, resulting in the distribution observed in Figure 1. Thepattern in this longitudinal section of the kidney can now bereconciled with the distribution observed in the transversesections of the kidney in Figure 2. These anatomical features ofthe inner stripe of the outer medulla may explain the absence ofsilver grains in the vasa recta from the electron microscopicautoradiographs. However, the technique used in the present

study does not exclude the possibility of low affinity bindingsites on the vasa recta or tubular components, since theradioligand will be lost from low affinity sites during thewashing procedures. Nevertheless, tissue sections for the lowresolution receptor autoradiography are also subject to washingfollowing incubation, but the intense binding in the inner stripeof the outer medulla persists (Fig. 1). This explanation isconsistent with our previous observations of high-affinity Ang IIreceptors in the inner stripe using autoradiography [14, 181 andin membrane fractions prepared from rat renal outer medulla[13], as well as the report of high affinity binding in rabbitcultured renomedullary interstitial cells [161.

The cells in the interstitium of the inner stripe of the outermedulla that bind '251-[Sar1, 11e8] Ang II could be identified astype 1 interstitial cells [19]. In the rat kidney, type 1 interstitialcells predominate in the inner stripe of the outer medulla andare the only type of interstitial cells in the inner medulla [20]. Inthe present study, at the resolution of the electron microscope,the interstitial cells bearing Ang II binding sites share morpho-logical characteristics of type 1 interstitial cells, including longcytoplasmic projections, cisternae of rough endoplasmic retic-ulum, free ribosomes, lysosomes and vacuoles in the cytoplasmalong with small bundles of filaments. However, only a few lipiddroplets were observed in these cells in our study. The currentfinding of binding of '251-[Sar', lie8] Ang II to type 1 interstitialcells in the inner stripe, but not to type I cells of the innermedulla [13—15], probably indicates two subtypes of type Iinterstitial cells. Differences in the biological activities of type 1cells have been reported previously between the outer medullaand the inner medulla [21, 22].

Previous studies suggested that the type 3 interstitial cell, apericyte with contractile elements, was a possible site on whichAng II acts to modulate medullary and papillary microcircula-tion [4, 5, 12]. In the present study, however, neither the type3 nor the type 2 interstitial cell, a lymphocyte-like cell ormacrophage [23], were found to bear a high density of Ang IIbinding sites. The type 2 cell is distributed throughout thecortex, the outer medulla and the outer part of the inner medulla[20], whereas the type 3 cells are mainly located in the outermedulla and the outer part of the inner medulla [24]. Thedistribution of these two other types of interstitial cells in thekidney does not correlate well with the patterns of anatomicaldistribution of renal Ang II receptors, as revealed by in vitroautoradiography [13—151.

The physiological significance of the occurrence of Ang IIreceptors on type 1 interstitial cells of the inner stripe of theouter medulla remains to be elucidated, as the function of thesecells are currently not known. Early reports suggested that theyprovide structural support in the medulla [25] because they areoften arranged in rows between the loops of Henle and thedescending vasa recta, with their long axis perpendicular to thatof adjacent tubules and blood vessels permitting a single cell tobe in close contact with several vessels and the tubules [201.However, the functions of the type 1 interstitial cells appear tobe more complex than a supporting action. Although it isunlikely that the type 1 cells can directly constrict the bloodvessels, they have bundles of microfilaments located at theirperiphery [20], as do the mesangial cells. Mesangial cells haverecently been likened to pericytes [26], with multiple functionsincluding contraction, structural support, phagocytosis and

1378 Zhuo et al: Ang II receptors in rat renal medulla

Fig. 5. Light and electron micrographs of the inner stripe of the outer medulla of the rat kidney incubated with 260 M '251-[Sar', Ile8j Ang II inthe presence of an excess (1 i)ofunlabelled[Sar', lIe8] Ang II for one hour. A. Dark field illumination demonstrating non-specific backgroundof grain development across the tissue section (T) when compared with the background (B). Magnification: x 100. B. Dark field illumination athigher resolution showing that grain development does not follow the outline of the tubules or vasa recta bundle components when compared withFig. 2D, and is limited to non-specific background levels. Magnification: x400. C. Electron micrograph of interstitial cells from a vasa recta bundleshowing non-specific grain development. Abbreviations are: TL, thin limb; IC, interstitial cell; L, lipid inclusion. Stain: uranyl acetate, lead citrate.Magnification: x4,500.

generation of vasoactive agents [27]. It has recently been rate. Thus, in a manner similar to the mesangial cells, the typeproposed that contraction of the mesangial cells may affect 1 interstitial cells might influence blood vessels or the tubulesglomerular capillary geometry and hence glomerular filtration via their connections with basement membranes of these struc-

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Zhuo et al: Ang II receptors in rat renal medulla 1379

tures. In the medulla, interstitial Ang II could act on interstitialcells to alter their size and shape and thereby those of theadjacent vasa recta, consequently modulating the medullarymicrocirculation and countercurrent exchange mechanisms.This proposal is supported by reports in which Ang II causesreduction in vasa recta and papillary blood flow, and blockadeof converting enzyme produces opposite effects [4, 6]. How-ever, it is not known whether these effects are mediated by thedirect contraction by Ang II of the descending vasa recta orcapillaries, or by Ang II receptors distributed in the type 1interstitial cells of the inner stripe of the outer medulla. In thepresent study, we found no binding on the vasa recta bundles,or other small blood vessels, thus providing no support for adirect contraction. A similar conclusion came from the study byCupples, Sakai and Marsh [8] who found that exogenous AngII, delivered systemically, failed to alter the vasa recta bloodflow and vasa recta diameter. Recently, we have observed thatAng II increases papillary blood flow, perhaps secondary tokinin release [281.

Localization of Ang II receptors in the type 1 interstitial cellsof the inner stripe also suggests a role for interstitial Ang II inregulating renal medullary function. It is well documented thatAng II can be generated intrarenally [29, 30] and that Ang IIconcentrations in the kidney tissues [31, 32] and in fluidssampled from different compartments of the rat kidney are wellabove those in plasma [33]. In addition, the renal medulla is oneof major sites where prostaglandin formation takes place in thekidney [34]. Ang II is known to stimulate phosphatidylinositoland polyphosphoinositide turnover in the rat renal medulla [101.Ang II receptors in cultured renomedullary interstitial cellshave been linked to the production of prostaglandin in thesecells [9, 161. Interaction between Ang II and prostaglandins mayplay an important role in regulating medullary blood flow [8].Since interstitial hypertonicity in the renal medulla is vital forurinary concentrating and diluting processes, local Ang II maymodulate these mechanisms by other unidentified actions, suchas modulation of active sodium transport in the thick ascendinglimb of the loop of Henle to preserve interstitial hypertonicity inthe renal medulla. Localization of Ang II receptors to the type1 interstitial cells of the inner stripe of the outer medullaprovides support for such a medullary action of the peptide orother local paracrine actions on vascular or tubular function inthe medulla.

In summary, the present findings from high resolution elec-tron microscopic autoradiography indicate that a subgroup oftype 1 interstitial cells are the major sites for the high density ofAng II receptors located in the inner stripe of the outer medulla,and suggest that intrarenal Ang II may interact with these cellsto modulate medullary microcirculation or tubular function inthe medulla either by their contractile function or by localparacrine actions.

Acknowledgments

This work was supported by grants from the National Health andMedical Research Council of Australia, National Heart Foundation ofAustralia and Australian Kidney Foundation. The technical expertise ofMs. Jane McCausland is greatly appreciated.

Reprint requests to Professor F.A.O. Mendelsohn, Department ofMedicine, University of Melbourne, Austin Hospital, Heidelberg 3084,Victoria, Australia.

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