0022-1554/921$3.30 The J o u d of Histochemistry and Cytochemhy Copyright 0 1992 by The Histochemical Society, Inc

Vol. 40, No. 11. pp. 1685-1691, 1992 Printed in UXA.

Original Article

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Demonstration of Dopamine-immunoreactiveCells in the Proximal Convoluted Tubule of Gerbil (Metiones unguiculatus) Kidney RALPH R. DAWIRS' and GERTRAUD TEUCHERT-NOODT Department of Neuroanatomy, Faculty of Biology, University of Bielefeld, Bielefeld, Gemany. Received for publication February 21, 1992 and in revised form May 26, 1992; accepted June 9, 1992 (2A2606).

We investigated dopamine immunoreactivity in the kidney of gerbils (Meriones unguiculatus). For that purpose a sensitive and selective antibody against glutaraldehyde-conjugated dopamine was applied. Dopamine-immunoreactive cells were found in the epithelium of the proximal convoluted tubule, where these cells revealed a typical segment-likedistribution pattem. Dopamine-i"unoreactive precipitateswere small and concentrated at the apical pole of the labeled cells.

Introduction As in the central nervous system, where it has long been established that dopamine (DA) acts as as a true neurotransmitter (Carlson, 1987), evidence is accumulating that in the peripheral nervous system also DA might function as a neurotransmitter in its own right, rather than just being a precursor of norepinephrine (NE) and epinephrine (Bell, 1988; Lackovic and Neff, 1983; Bell, 1982a; Bell and Lang, 1982). From pharmacological studies, DA and NE are assumed to be located at separate nerve endings (Neff et al., 1986), with DA acting via specific DA receptors in various peripheral tissues (Marzinoet al., 1986; Missale et al., 1986; Ruffalo and Morgan, 1984; Valenzuela and h i , 1982; Schnurkes and Van Nueten, 1981; Lanfranchi et al., 1978). The peripheral functions of DA are characterized by a variety of effects, influencing blood pressure (Neff et al., 1986),modulating autonomic neuromuscular transmission (Bell and Lang, 1982), stimulating pancreatic secretion (Hashimoto et al., 1973),and controlling motility of the gastrointestinal tract (Marzino et al., 1990). To perform these functions, DA has direct effects on several smooth muscles and neurons (Grivegnee et al., 1984; Ruffalo and Morgan, 1984; Furness and Costa, 1982; Sahyoun et al., 1982) and may also act indirectly, modulating tissue hormone levels (Di Giulio et al., 1989) and presynaptic release of other neurotransmitters(Kusunoki et al., 1985; Ruffalo and Morgan, 1984).

Correspondence tO: Dr. Ralph R. Dawirs, Univ. of Bielefeld, Faculty of Biology, W-4800 Bielefeld, Germany.

This study has directly identified dopamine as a constituent of certain ceh of the proximal convoluted tubule in gerbils. The functional significanceof dopamine in these cells is discussed in relation to the present view of renal dopaminergic actions. (JHistochem Cytochem 40:1685-1691, 1992) KEYWORDS: Dopamine; Immunocytochemistry;Kidney; Proximal convoluted tubule; Extraneural dopamine; Gerbil.

In the kidney DA promotes renal blood flow (RBF), provoking vasodilation (Bell and Lang, 1982,1973; Chapman et al., 1982; Dinerstein et al., 1979; Goldberg et al., 1978)and further increasing the glomerularfiltrationrate (GFR) (Baines and Drangova, 1986; Pelayo et al., 1983; Goldberg et al., 1978) and renin release (Bell and Lang, 1978; Imbs et al., 1975). DA is generally considered to function as a physiologically significant natriuretic factor (Ball et al., 1978; Kim et al., 1980; Huges et al., 1988;Jose et al., 1986; Krishua et al., 1985; Cuche et al., 1983; Goldberg and Weber, 1981; Baines and Morganov, 1980; Boren et al., 1980; McGrath et al., 1980), controlling the normal salt and water balance in cooperation with anti-natriuretic-acting NE (Ball et al., 1982; Stephenson et al., 1982; Di Bona, 1977). In this connection, locally formed DA was found to inhibit the Na+,K+pump in proximal convoluted tubule (PCT) cells (Bertorello et al., 1988; Aperia et al., 1987). Although renal nerves containing DA have as yet not been directly demonstrated,indirect evidence, mostly obtained by fluorescence histochemistry and from pharmacological experiments, indicates that DAergic nerve terminals may be present in the mammalian kidney (Bell, 1988,198213; Dinerstein et al., 1983; Lackovic and N d , 1983; Morgunov and Baines, 1981; Dinerstein et al., 1979), predominantly innervating the outer renal cortex (Bell et al., 1978; Dinerstein et al., 1979; Bell and Gillespie, 1981). Until recently, increasing evidence speaks for additional extraneural sources of renal DA (Adam and Adams, 1985; Baines, 1982; Ball et al., 1982; Stephenson et al., 1982; Baines and Chang, 1980) which should be primarily produced in PCT cells (Baines et al., 1985; Hagege and Richet, 1985;Hagege et al., 1985; Baines and Drangova, 1984; Wahbe et al., 1982; Baines and Chang, 1980), where the concen1685

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tration of dopa decarboxylase, the enzyme that converts dopa to DA, is high (Goldstein et al., 1972). This non-neural DA was assumed to act as a paracrine substance at tubular DA-1 receptors to decrease sodium transport (Bertorello et al., 1988; Seri et al., 1988; Siragy et al., 1988; Aperia et al., 1987;Jose et al., 1986; Sowers et al., 1984; Pelayo et al., 1983; Unger et al., 1978; Ball and Lee, 1977). However, as in the case of DA-containingrenal nerves, DAergic non-neural cells in the kidney have as yet not been directly demonstrated. In the present study we therefore investigatedDA immunoreactivity in the kidney of gerbils (Meriones unguiculatus) with a specific antibody against glutaraldehyde-conjugatedDA. Results are discussed against the background of current understanding of renal DA function.

Materials and Methods Preparation of Tissue. Gerbils (Merzones unguiculatus) of both sexes were kept under natural daylnight cycles in home cages (30 x 40 cm) with two animals each. Food and water were provided ad libitum. Adult gerbils (n = 7) were transcardically perfused under deep chloral hydrate anesthesia (1.7 glkg, IP). The blood was washed out with approximately 60 ml cold oxygen-enriched 0.05 M phosphate buffer (pH 6 . 2 ) containing 1% sodium metabisulfite, followed by 500 ml 5% glutaraldehyde with 1% sodium metabisulfite in 0.1 M phosphate buffer (pH 7.6). Finally, the fixative was quickly washed out with 0.05 M Tris-buffered saline (TBS) containing 1%sodium metabisulfite (pH 7.5; wash buffer). Immediately after perfusion the kidneys were dissected and 40-pm thick cross-sections performed with a vibratome (Model G, Campden Instruments, London, UK), which were collected in wash buffer at 4°C. Immunocytochemical Procedures. For light microscopy (Reichert-Jung, Polyvar), the peroxidase-anti-peroxidase (PAP) method (Sternberger, 1979) was the chosen immunocytochemical procedure, which was performed freefloating with the following incubations: (a) pre-incubation in 0.5% H202 (10 min, 4’C) to eliminate endogenous peroxidase activity; (b)pre-incubation in 10% normal goat serum (NGS) (Sigma; Deisenhofen,Germany) in wash buffer (0.5 hr, 4°C) to reduce nonspecific background staining; (c) rabbit antiserum against glutaraldehyde-conjugated DA (Incstar; Stillwater, MN) diluted 1:lOOO in wash buffer containing 0.4% Triton X-100 and 1%NGS (16 hr, 4’C) to improve antibody penetration. This highly specific and sensitive antibody against DA was first obtained and characterized by Geffard et al. (1984). Specificity controls included radioimmunological assays and incubations with catecholamine-covered Sepharose beads to exclude cross reaction (Buijs et al., 1984); (d) three 10-min washes in wash buffer; (e) goat anti-rabbit serum (GAR) (Sigma),diluted 1:50 in TBS containing0.4% Triton X-100 and 1% NGS (2 hr, room temperature);(f) three lO-min washes; (g) peroxidase-anti-peroxidase (PAP) (Sigma) in the same dilution buffer as for GAR, 1:500 (2 hr, room temperature); (h) three 10-min washes; (i) o.O>% 3,3’-diaminobenzidine(DAB) (Sigma) and 0.01% H202 in TBS containing 2% (NH&S04NiS04 (15 min. room temperature); (j) three 10min washes; (k) sections were dried on glass slides overnight, dehydrated, mounted in DePeX (Serva; Heidelberg, Germany), and coverslipped. Control sectionswere treated as described above, but the primary antibody was omitted or pre-treated with DA conjugated to bovine serum albumin with glutaraldehyde, the same antigen used to generate the antibody. No immunolabeled cells were seen in either case. Although the antibody serum used reveals some nonspecific affinity to connective tissues, specific locations were nevertheless easily identified in epithelial cells with the above-described procedure.

Preparation for Electron Microscopy. The immunostaining procedure

DAWRS, TEUCHERl-NOODT

described above was particularly adapted for electron microscopy. No Triton X-100 was added to the sera used. Antiserum against glutaraldehydeconjugated DA (Incstar) was applied over 42 hr at 4°C (c), and both GAR and PAP (Sigma) were added for 2 hr at 37°C (e,g). DAB (Sigma) was applied without (N&)2S04NiS04 (i), After three 10-min washes (j) sections were incubated in 2% os04 for 1 hr. The slices were finally washed, dehydrated, and flat-embedded in Epon (Serva). Semi-thin sections were provided to detect immunolabeled cells by phase-contrast microscopy (Zeiss; Oberkochen, Germany) before ultra-thin sections for electron microscopic analysis (Hitachi; Tokyo,Japan). Controls were treated as described for light microscopy.

Results DA immunoreactivitywas found in epithelial cells of the proximal convoluted tubule (PCT) along certain segments (Figure la). Although the typical brush border and vacuoles of the apical endocytotic apparatus appeared artificially altered in histological examination of whole slices, these cells could unequivocally be identified as cells of the PCT. DA immunoreactivity was concentrated around the apical membrane of these cells (Figures 1b and IC).Two labeled PCT cells identified by phase-contrastmicroscopy (Figure IC)are shown in the electron micrographsof Figure 2. Noncontrasted (Figure 2a) and subsequent ultra-thin slices contrasted with uranyl acetate and lead citrate (Figure 2b) were compared to localize DA immunoreactivity. Small DA-immunoreactive electrondense precipitates concentrated between the nucleus and the apical membrane of these cells were identified in non-contrastedslices (Figure 2a) and could be recovered in subsequent contrasted slices (Figure 2b).

Discussion The present study has demonstrated dopamine (DA) immunoreactivity in cells of the proximal convoluted tubule (PCT) in gerbils. Although until recently renal DA was thought to originate principally from nerve terminals (Bell, 1982a,b),it is currently believed that additional extraneural sources of DA should exist in the mammalian kidney (Adam and Adams, 1985; Ball et al., 1982; Baines and Chang, 1980). After a number of indications suggesting that cells of the PCT produce and handle non-neural renal DA (Jose et al., 1986; Hagege et al., 1985; Baines and Chang, 1980; Goldstein et al., 1972),this study has directly identified DA in these cells. The existence of DA-containingPCT cells is in good accordance with early and recent findings on renal DA functions. Since urinary DA occurs in much higher concentrations than could be expected by filtration of free plasma DA from arterial blood, it has been proposed that DA is produced in the kidney (Lackovic et al., 1981; Ball et al., 1978). In isolated perfused kidneys, urinary DA excretion was abolished by pre-treatment with a dopa decarboxylase inhibitor, and was increased by L-dopa but not by DA conjugates in the perfusate (Adam and Adams, 1985). In incubated kidney slices, DA production depends on L-dopa in the medium (Hagege et al., 1985). Therefore, it seems likely that most if not all renal DA comes from decarboxylation of circulating L-dopa rather than from free plasma DA or DA conjugates (Sowers et al., 1984; Baines and Chang, 1980; Ball and Lee, 1977). Renal DA excreted into both the urine and the circulation could be released from re-

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Figure 1. (a) Distribution of DA-immunoreactive cells in proximal convoluted tubules of gerbils. (b.c) DA immunoreactivity is marked by arrows in some labeled cells. PCT, proximal convoluted tubule; DT, distal tubule. (c) Two cells marked by asterisks are shown in electron micrographs in Figure 2. Original magnifications: a x 400; b.c x 1000. Bars: a = 50 pm; b,c = 5 pm.

nal nerves or from non-neural cells (Ball et al., 1982; Lee, 1982; Stephenson et al., 1982). Nevertheless, neither renal DA production nor urinary DA excretion was diminished after prior denervation of :he kidney (Adam and Adams, 1985; Hagege et al., 1985; Baines and Chang, 1980), indicating that DA production must have occurred in extraneural tissue. In the kidney, L-aromatic amino acid decarboxylase(AADC). the enzyme that forms DA from L-dopa, is abundant and is primarily, if not exclusively, located in cells of the PCT (Baines and Chang, 1980; Goldstein et al., 1972). The segment-like appearance of AADC-positive immunofluorescence in the PCT (Bertorello et al., 1988) tallies with findings that L-dopa-induced DA histofluorescence, strictly limited to the PCT, occurred only in certain segments of the tubule (Hagege and Richet, 1985; Hagege et al.. 1985; Wahbe

et al., 1982). Furthermore, both L-dopa-induced fluorescence and AADC-positive immunofluorescence were mainly located at the apical pole of the PCT cells (Bertorello et al., 1988; Hagege et al., 1985). All this corresponds well with our results that DA immunoreactivity was found exclusively in cells of the PCT along certain segments, with immunoreactive granules concentrated around the apical membrane of these cells. The present identification of D.4-containing PCT cells may support current concepts on how renal DA might realize its natriuretic influence. In PCT segments incubated with L-dopa or DA, the Na+,K+-ATPasewas reversibly inhibited, leading to increased sodium excretion (Aperia et al., 1987). There is evidence that inhibition of renal Na',K+ activity might be regulated via DA-1 receptormediated stimulation of protein kinase C activity in PCT cells

PI

PCT

Figure 2. Electron microscopic identification of typical DA-containingcells of the proximal convoluted tubule (PCT) of gerbils. (a) Electron-dense PAP complexes marked by arrows appear between areas marked by solid lines. Identical asterisks mark comparable areas in (a) noncontrastedand (b)contrasted slices. Nucleus (N). Original magnification x 10,000. Bar = 2 pm.

DOPAMINERGIC CEUS IN THE KIDNEY

(Kinoshita et al., 1990; Bertorello and Aperia, 1989). In addition, it has been proposed that DA might act by a mechanism independent of DA receptors, inhibiting sodium reabsorption by DA formed intracellularly (Aperia et al., 1987). This means that locally produced DA may act as a paracrine substance to control sodium reabsorption in those tubular segments that contain dopa decarboxylase, but also in more distal segments of the nephron (Huges et al., 1988; Aperia et al., 1987; Bello-Reuss et al., 1982). From morphological and pharmacological investigations it has been concluded that this paracrine natriuretic function of DA-containingPCT cells should be under direct sympathetic control (Lee, 1982; for recent discussion see Baines and Drangova, 1986), most likely mediated via noradrenergic innervation of these cells (Di Bona, 1977). Therefore, the physiological balance between natriuretic (DAergic) and anti-natriuretic (noradrenergic) stimuli that control the sodium transport process in tubular cells may be understood as noradrenergic neurogenic inhibition of DAergic paracrine function. In this study we did not see any DA immunoreactivityin nerve terminals of the kidney. This seems contrary to evidence indicating the presence of DA-containing neural elements in the kidney (e.g., Bell, 1988,1982a; Baines and Drangova, 1986; Dinerstein et al., 1983; Lackovic and Neff, 1983). It may be argued that the immunocytochemical procedure applied in this study might be sensitive for non-neural but less sensitive for neural tissues. However, in parallel experiments using exactly the same procedure with antibodiesfrom identical stocks, DAergic neurons were stained in gerbil brain (Dawirs et al., 1991),whereas no DA-containing nerves could be labeled in the gerbil kidney. Still, we would not totally deny that DA-containingand functionallyimportant axon terminals were present, since acute DA levels might be below some detectable concentration while turnover rates might be high. In any case, direct demonstration of DA-containing renal nerves is still not available. Hence, it has been argued that ifa DAergic innervation of the kidney was present it should be minor compared with the large amount of DA produced at extraneural sites (Adam and Adams, 1985). Although DA-1 and DA-2 receptors were found in various renal structures and their selective sensitivity against DAergic agonists and antagonists was shown to be related to well-known DAergic functions in the kidney (e.g., Felder et al., 1989; Katayama et al., 1989; Siragy et al., 1988;Jose et al., 1986), the functional significance of renal DA-containingnerves remains unclear. It has been recently reviewed that the relation of proposed DAergic nerves to DA receptors is unknown, since it has not been thus far demonstrated that alterations in renal functions induced by reflex or electrical activation of efferent renal nerves could be inhibited by specific and selective DA receptor antagonists (Di Bona, 1990). Therefore, we might say that the fact that there is no proof of functional significance of renal DA-containingnerves is consistent with our negative results on DA immunoreactivity in renal nerves. Nevertheless, this issue seems far from being settled and should be the subject of future investigations. In conclusion, the presence of DA-immunoreactive cells in the PCT of gerbil kidney confirms earlier assumptions from indirect evidence that substantial quantities of renal DA should be synthesized at extraneural sites. It would be of particular interest to analyze, both structurally and histochemically, sympathetic innervation of these DAergic tubular cells to further evaluate mechanisms

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of mutual functions of NE and DA in regulating plasma salt and water balance.

Acknowledgments We aregratefulto Mr E. Kemming-GmbnerandMr U. Schmederfortechnicdassistance. We shouldlike t o than&Ms E. firvouni, who he(pedwith some experiments and histologicdpreparations. We are also indebted to Mr K. Weigel, who he(pedwith the photographic kayout. Our specialthanh are due to Mr Fay MirselbrooA for correcting the English drafi

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