Cell Tissue Res (1995) 281 :197-206

Cell&Tissue Research 9 Springer-Verlag 1995

In situ hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys Ian A. Darby 1, *, Conrad Sernia 2

1Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Victoria, 3052, Australia 2 Department of Physiology and Pharmacology, University of Queensland, St. Lucia, 4072, Australia Received: 29 November 1994 / Accepted: 1 February 1995 Abstract. Recent evidence suggests that a local reninangiotensin system is operational in the kidney and that it mediates some of the actions of angiotensin II on renal tubules. In this study the ontogeny and renal distribution of the unique precursor to angiotensin lI formation, angiotensinogen, was investigated in rats by use of immunohistochemistry, immuno-electron microscopy and non-isotopic hybridization histochemistry. At the lightmicroscopic level, intense staining for angiotensinogen was found in the proximal convoluted tubules of the cortex, with lighter staining in the straight proximal tubules of the outer stripe. The strongest immunostaining was found in the kidneys of neonatal rats, where glomerular mesangial ceils and medullary vascular bundles were also immunopositive. The angiotensinogen content of the kidneys in late gestation embryos and neonates showed the presence of angiotensinogen by day El8 and a peak content in the neonate. Non-isotopic hybridization histochemistry with biotinylated oligodeoxynucleotide probes confirmed the presence of angiotensinogen mRNA expression in the proximal convoluted tubules of the renal cortex. Electron-microscopic immunohistochemistry showed staining of relatively few electrondense structures close to the apical membrane of proximal convoluted tubule cells in the adult kidney. In the neonatal rat kidney, angiotensinogen immunostaining at the electron-microscopic level was found throughout the proximal tubule cells and was markedly stronger than that seen in adult kidney. The presence of angiotensinogen, from embryonic day 18, in the proximal tubules, mesangial cells and vasculature of the kidney suggests multiple potential sites of intrarenal angiotensin II generation with an ontogeny in late gestation.

* Present address and address for correspondence: Wound Foundation of Australia, Heidelberg Hospital, Heidelberg West, Victoria, 308l, Australia

Key words: Renin-angiotensin system - Morphology Renal tubules - Ontogeny - Rat (Wistar)

Introduction

There is considerable evidence for an extravascular production of angiotensin II (Ang II) (Fei et al, 1981; Phillips et al. 1993). A likely site for such an extravascular renin-angiotensin system is the kidney, where local Ang II appears to feature in sodium reabsorption, tubuloglomerular feedback and inhibition of renin secretion (Harris and Young 1977; Naftilan and Oparil 1978; Navar and Rosivall 1984; Harris and Navar 1985; Mitchell and Navar 1991). An additional, and perhaps more significant role of local Ang II lies in its mitogenic action, partly through stimulation of transforming growth factor-~ (Wolf et al. 1993). Via this mitogenic role, renal Ang II becomes a relevant factor in the compensatory hypertrophy which often follows renal disease, as well as in the normal embryonic development of the kidney (Friberg 1994; Norman. 1991). The major site of Ang II action on sodium reabsorption is the proximal convoluted tubule and there is evidence that it may also be a site of Ang II production. Ang II appears to be released into, or formed in the lumen of the proximal convoluted tubule where Ang II concentrations are markedly higher than plasma levels and increase along the proximal tubule length (Seikaly et al. 1990; Braam et al. 1993). The unique substrate for the generation of Ang II is angiotensinogen and the probable site for its production is the cortical proximal tubule which has been reported to contain angiotensinogen mRNA (Ingelfinger et al. 1990). In the present study we have combined light- and electron-microscopic immunohistochemistry to localize angiotensinogen at the cellular and ultrastructural levels, and in situ hybridization with biotinylated oligonucleotides to visualize cellular angiotensinogen mRNA. In addition, we have examined the ontogeny of angiotensinogen in order to deter-

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Fig. 1A-C. Localization of angiotensinogen in adult rat kidney by immunohistochemistry with alkaline phosphatase and nitro blue tetrazolium - 5-bromo-4-chloro-3indolyl phosphate as chromogen. A Intense staining of cortical proximal convoluted tubules showing cytoplasmic distribution skewed towards lumen of tubule. In the adult, the glomerulus (GLOM) was usually free of stain. B Control section in which the primary antibody had been pre-incubated with angiotensinogen-rich plasma from nephrectomized rats. The same results were obtained by incubation with 1 mg/ml of pure angiotensinogen. C Staining of convoluted (P*) and straight (P) segments of proximal tubules in region of outer stripe of kidney. Collecting ducts (CD) unstained. x300

199 mine the earliest embryonic stage at which the local generation of A n g II b e c o m e s possible.

Materials and methods Outbred Wistar rats from the Central Breeding Houses at the Universities of Melbourne and Queeensland were used in all experiments. A total of 10 male and 10 female adult rats and fetal tissues from rats of known gestation stage were used. Animals were killed with an overdose of barbiturate anesthesia. Tissues for light microscopy were obtained from rats perfused intracardially with 4% paraformaldehyde, post-fixed, embedded in paraffin and processed for immunohistochemistry as described previously (Thomas and Sernia 1988). Sections (5 btm thick) were incubated in polyclonal antibodies raised against purified rat angiotensinogen at a dilution of 1:2000. Detection of immunostaining was performed by use of a standard alkaline phosphatase reaction with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate as the chromogen, and 1 mM levamisole as an inhibitor of endogenous alkaline phosphatase. For immuno-electron microscopy, small blocks of renal cortical kidney were fixed overnight in 2% paraformaldehyde and 0.1% glutaraldehyde, then dehydrated through graded ethanols, embedded in LR White (London Resin Co. Ltd. Basingstoke, UK) embedding resin and polymerized for 48 h at 40~ The primary antibody was applied to sections at 1:500 dilution overnight at 4~ and detection of specific staining was performed with a goat anti-rabbit IgG secondary antibody conjugated to 1 nm gold (Amersham, Buckinghamshire, UK) diluted 1:100. Silver enhancement of staining was performed for 4-5 min with IntenseM (Amersham, Buckinghamshire, UK). Negative controls for both light-microscopic and electron-microscopic immunohistochemistry included incubation in pre-immune serum and preabsorption of the primary antibody with purified rat angiotensinogen. Non-radioactive in situ hybridization histochemistry was performed on sections from rat kidneys embedded in paraffin after overnight fixation in 4% paraformaldehyde. Protease treatment of sections was performed by incubation with 0.IN HC1 for 15 min followed by proteinase K 50 mg/ml for 30 min at 37~ (Sigma, St. Louis, Mo., USA). Sections were then washed in 50 mM TRIS-buffered saline containing 0.5% Tween 20. Sections were hybridized overnight at 40~ in Rapid-Hyb buffer (Amersham; Buckinghamshire, UK) containing a mixture of two biotinylated antisense oligonucleotide probes at a concentration of 400 ng/ml each. The sequences of these probes are complementary to positions -40 to -1 and 73 to 114, respectively, of the angiotensinogen mRNA sequence (Ohkubo et al. 1983). The oligonucleotides were synthesized by the Centre For Molecular Biology at the University of Queensland and biotinylated during synthesis with a phosphoroamidite reagent (Biotin-ON; Clontech, Palo Alto, Calif., USA) by adding biotin at the 5' end and at the 10th, 20th and 30th nucleotide positions. Biotinylated probes were purified by high pressure liquid chromatography. Following the overnight hybridization, sections were washed sequentially in 5xSSC, 2xSSC and 0.5xSSC and the biotin on the hybridized probes was detected by the same alkaline phosphatase method used for immunohistochemistry. As a negative control, sections were pretreated with RNase A (Sigma, St. Louis, Mo., USA) at a concentration of 1 mg/ml for 30 rains prior to hybridization with the biotinylated oligonucleotide probes. In situ hybridization histochemistry of rat liver sections was used as a positive control. The kidney content of angiotensinogen was measured at gestational days 16, 18 and 20; in neonates and adult mate and female rats. Whole kidneys were homogenised in 1:9 (weight:volume) of H20 containing 10 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (PMSF) to inhibit proteases and the homogenate centrifuged at 10 000xg for 30 min. The supernatant was adjusted to pH 4.5 with acetic acid, left at 4~ for 1 h, and then centrifuged at 1000xg for 20 min. This procedure effectively removes peptid-

ases (Sernia and Mowchanuk 1983). The angiotensinogen in the supernatant was measured by a direct radioimmunoassay, as described previously (Thomas et al. 1992). Embryonic kidneys were divided into three pools, each containing 10-12 kidneys. In adults, angiotensinogen was measured in each of six kidneys.

Results Light-microscopic immunohistochemistry of adult and neonatal kidneys showed the most intense staining in the cortex (Fig. 1-2). In the adult kidney the cortical proximal convoluted tubule cytoplasm was the most conspicuously stained, followed by staining o f lower intensity in the straight and convoluted proximal tubules in the outer stripe of the medulla (Fig. 1C). In the adult proximal convoluted tubule, the stain was not evenly distributed in the cytoplasm but was frequently skewed towards the apical surface (Fig. 1A). Staining was not seen in the mesangial, juxtaglomerular or macula densa cells o f the adult kidney. The neonatal kidney stained more intensely than the adult kidney (Fig. 2). The stain was localized in the maturing cortical tubules except for light cytoplasmic staining in tubule bundles of the inner medulla and intense staining in capillary endothelial cells (Fig. 2C). Unlike the adult kidney, glomerular mesangial cells showed intense immunostaining (Fig. 2D). The renal arterial vasculature, f r o m the larger arcuate arteries to the afferent arterioles, stained for angiotensinogen (Fig. 3). The staining was localized to the vascular smooth muscle. Veins and lymphatic vessels did not stain. No staining was seen in kidney sections w h e n pre-immune serum was substituted for the angiotensinogen antibody (result not shown). In situ hybridization histochemistry o f adult kidney with biotinylated oligonucleotide probes to angiotensinogen m R N A is shown in Fig. 4. As found with immunohistochemistry, the angiotensinogen m R N A was localized in convoluted proximal tubules and, at much lower levels, in proximal tubules in the outer stripe of the medulla. The nuclei were clear of stain and its distribution in the perinuclear cytoplasm was even. Evidence of staining in mesangial cells and inner medullary tubules was not found. Sections in which m R N A was digested with RNase A showed no specific hybridization. In situ hybridization histochemistry of neonatal kidney was not attempted. The difference in the intensity of i m m u n o h i s t o c h e m istry stain between neonatal and adult kidney was confirmed by measurements of kidney angiotensinogen content (Fig. 5). Kidney h o m o g e n a t e s from rats at various ages s h o w e d the presence o f 1.5_+0.05 m g angiotensi-

)

Fig. 2A-D. Immunohistochemical localization of angiotensinogen in neonatal rat kidney. A Low-power (x15) view of neonatal kidneys showing distribution of blue immunostain in developing cortex and medulla. Boxed letters correspond to areas shown in higher power micrographs, B, D x156 and x235 magnification of cortex showing staining in proximal tubules and in mesangial cells of glomeruli (arrowheads in D). C Medullary region of kidney showing intense staining in capillary vessels and absent or faint staining in tubules, x235

200

Fig. 2A-D

201

Fig. 3A-D

202

Fig. 4A-D. In situ hybridization histochemistry of adult rat kidney with biotinylated antisense probes to angiotensinogen mRNA. Hybridized probe visualized with a streptavidin-biotin-alkaline phosphatase complex and nitro blue tetrazolium and 5-bromo-4chloro-3-indolyl phosphate substrate. A, C Lower power (xl00) micrographs of cortex and medulla, respectively. B Higher power nogen/mg protein by embryonic day 18, followed by rapid increases to 8.49+_0.45 mg angiotensinogen/mg protein in the neonatal kidney. The content in adult kidney was lower than in neonates, and a higher content was found in females (3.98_+0.44 mg angiotensinogen/mg protein) than males (1.0+__0.2 mg angiotensinogen/mg protein). Electron-microscopic immunohistochemistry showed specific staining, with gold grains localized in electrondense granules close to the apical membrane of the proxi-

(x300) view of cortex showing angiotensinogen mRNA in proximal tubules and not in glomeruli. D Higher power (x300) of boxed inset in C showing tubular staining in outer stripe of medulla. RNAse treatment abolished all staining (not shown). Note clear nuclei and intense perinuclear signal for angiotensinogen mRNA Kidney Angiotensinogen Content

10 N=3-6 Mean• tO

Female

(-

( ~ 4~ C~ 0 <

Male

(

Fig. 3. Immunohistochemical localization of angiotensinogen in (A) radial artery, (B) interlobular artery and afferent arteriole, and in (C) arcuate artery. D Control section with preabsorbed primary antiserum. Section stained lightly to limit extent of tubule stain. Veins and lymph vessels unstained, x160; x80 in B

N.D

E16 E18 E20 Neonate Adult Rats Fig. g. Content of angiotensinogen in adult, newborn and fetal rats. Angiotensinogen measured by direct radioimmunoassay. Not detectable (ND) at embryonic day 16

203

Fig. 6A, B. lmmuno-electron microscopy showing angiotensinogen staining in electron-dense granule (arrow) close to apical membrane in adult rat kidney proximal tubule cell. x20000. C Immuno-electron micrograph showing strong angiotensinogen stain-

ing of electron-dense granules (arrow) in proximal tubule cells of neonatal rat kidney close to apical membrane x12700. D Angiotensinogen immunostaining in neonatal rat kidney also located close to basal membrane, x 14600

real convoluted tubule cells in the adult rat kidney (Fig. 6). No other structures stained positively for angiotensinogen. In the neonatal rat kidney, immunostaining for angiotensinogen in proximal convoluted tubule cells was stronger and more widely distributed, with angiotensinogen-positive granules staining both in the apical part of the cell (Fig. 6c) and close to the basal membrane. (Fig. 6d) Pre-absorption of the antibody with purified rat angiotensinogen abolished staining (result not shown).

mesangial contractility and tubular sodium, hydrogen and bicarbonate ion transport (Harris and Young 1977; Naftilan and Oparil 1978; Navar and Rosivall 1984; Harris and Navar 1985; Mitchell and Navar 1991). It is also involved in renal growth, both directly and via stimulation of other growth factors (Wolf et al. 1993; Norman 1991). While some of these functions are mediated by angiotensin II in the systemic circulation,or by angiotensin II produced in the renal vasculature, there is persuasive evidence that renal tubules express the components of the renin-angiotensin system and thus potentially have the capacity to secrete angiotensin II or its precursors. The proximal convoluted tubule appears to be the major site of a tubular renin-angiotensin system. Thus angiotensin converting enzyme is present on the brush border

Discussion

Angiotensin II has multiple functions in the kidney as a regulator of renal haemodynamics, glomerular filtration,

204

of proximal convoluted tubule cells (Bruneval et al. 1986) and renin has been localized in these cells by immunocytochemistry (Taugner et al. 1979; Deschepper et al. 1986). Renin activity and renin mRNA have been found in proximal tubules, (Yanagawa et al. 1991; Moe et al. 1993; Chen et al. 1994) but its localization has yet to be confirmed by in situ hybridization. Angiotensinogen protein and mRNA expression have been reported in proximal convoluted tubule (Richoux et al. 1983; Ingelfinger et al. 1990; Terada et al. 1993), and may be regulated by Na (Ingelfinger et al. 1986) and by angiotensin II (Schunkert et al. 1992). There is also immunological evidence that angiotensinogen and renin are co-localized in the proximal convoluted tubule (Hunt et al. 1992). Finally, angiotensin II is present in proximal tubular fluid at concentrations 500-1000 times higher than those in plasma, and these high concentrations are maintained in the distal portions of the proximal tubules (Seikaly et al. 1990; Braam et al. 1993). The maintenance of high concentrations, in spite of a rapid metabolism of angiotensin II in the proximal tubule (Pullman et al. 1975; Carone and Peterson 1980), indicates that angiotensin II is produced from local angiotensinogen and renin, and is not residual angiotensin from glomerular filtration. In the present study, we have shown by immunohistochemistry that angiotensinogen is present in the proximal convoluted and straight segments of cortical and juxta-medullary nephrons, with the highest intensity in the cortical proximal convoluted tubule. This is in aggreement with in situ hybridization histochemistry with 32P-labelled probes, (Ingelfinger et al. 1990) and reverse transcription polymerase chain reaction of microdissected tubules (Terada et al. 1993). Our in situ hybridization histochemistry with biotinylated probes also localized angiotensinogen mRNA in the cytoplasm of proximal tubule cells in the cortex and the outer stripe, indicating that at least part of the angiotensinogen seen with immunohistochemistry by us and others (Taugner et al. 1979; Richoux et al, 1983; Hunt el al. 1992) is synthesized by the same cells. We did not consistently see evidence of angiotensinogen protein or mRNA in glomeruli, vasa recta or distal tubules of adult rats, as reported by others (Ingelfinger et al. 1990; Terada et al. 1993) but in neonatal kidneys intense staining of mesangial ceils and vascular bundles was observed. No staining was observed in granular cells of the juxtaglomerular apparatus, although it has been reported (Hunt et al. 1992). Hence there is now agreement between the various methods, immunohistochemistry, in situ hybridization histochemistry with radiolabelled and biotinylated probes, and polymerase chain reaction, that the proximal tubule is the major site of angiotensinogen expression, followed by lower levels of expression in mesangiat and medullary vascular bundles. This pattern of distribution correlates well with the localization of renin and Ang II receptors (AT 1 subtype) obtained by polymerase chain reaction of microdissected nephron segments (Terada et al. 1993; Chen et al. 1994). Renin mRNA has been detected only in the proximal convoluted and straight tubules (Chen et al. 1994), and the AT 1 mRNA has been found to be most abundant in proximal tubules, although all segments expressed the

transcript, as well as vasa recta (Terada et al. 1993). This distribution of receptors matches that observed by receptor autoradiography (Herblin et al. 1991) and is in accordance with evidence of angiotensin action downstream of the proximal tubule (Vander 1963; Zhuo et al. 1992). In addition to nephron components, arterial vessels, but not veins or lymphatic vessels, also stained intensely. Other components of the renin-angiotensin system are also present in renal vessels (Taugner et al. 1982; Tangner and Ganten 1982). Both conventional and electron-microscopic immunohistochemistry showed immunoreactive angiotensinogen to be more abundant in the neonate than in the adult. This pattern was confirmed by direct radioimmunoassay of kidney angiotensinogen at various ages. It is pertinent that in the adult, kidneys from female rats contained more angiotensinogen than those from males. In Wistar Kyoto rats the opposite has been reported by others, who have ascribed a higher angiotensinogen in males to regulation by testosterone (Ellison et al. 1989). A possible explanation could lie in the different strain of rat used. We could not detect angiotensinogen at embryonic day 16 but thereafter it increased rapidly and was high in the neonatal kidney. A high angiotensinogen content in the neonatal kidney has been reported previously (Gomez et al. 1988) and the ontogeny found in our study is similar to that of renin and the angiotensin receptors (TufroMcReddie et al. 1993). These rapid changes in the components of the renin-angiotensin system parallel the rapid growth in late gestation and in early neonatal life (Bogomolova 1966). It may therefore be speculated that the high activity of the renal renin-angiotensin system reflects a role for locally generated angiotensin II in the growth and differentiation of the developing kidney, in addition to its systemic homeostatic functions. Support for this speculation is found in the hypertrophic effect of angiotensin in mesangial and proximal convoluted tubule cells (Wolf et al. 1993). Studies with administration of angiotensin converting enzyme inhibitors and angiotensin receptor antagonists early in post-natal life have shown abnormal tubule development and impairment of renal function in rats (Friberg et al. 1994; McCausland et al. 1994). Hunt et al. (1992) colocalized renin and angiotensinogen to granule-like or lysosome-like structures spread throughout the cytopasm in both the kidney granular ceils and proximal convoluted tubule cells. We localized angiotensinogen in electron-dense vesicles at the apical and baso-lateral region in cells of the neonate and at the apical membrane in the adult kidney. The preponderance of immunoreactive angiotensinogen towards the tubular lumen was a distinctive feature of angiotensinogen staining in the proximal tubule of the adult (Fig. 1), which suggests some sort of trafficking across the apical membrane. There is a view, based on the absence of typical secretory granules in the proximal convoluted tubule and the capacity of these cells to take up large molecules, that the angiotensinogen staining in the proximal tubule represents pinocytotic degradation of exogenous angiotensinogen (Taugner et al. 1982; Richoux et al. 1983). While it is possible that the angiotensinogen in the prox-

205 imal tubule is part of a pinocytotic process from the glomerular filtrate, it is difficult to envisage appreciable amounts of it being available f r o m the glomerular filtrate of the adult rat, since the filtration coefficient for a protein close to the size o f albumin would be low. Alternatively, it m a y be postulated that angiotensinogen is synthesized by proximal convoluted tubule cells and used intracellularly to produce angiotensin I or II which is then secreted into the tubular lumen. In these circumstances, the angiotensinogen we detected in our ultrastuctural study would represent angiotensinogen or desA n g I-angiotensinogen in the process of degradation and not in the process o f exocytosis. This hypothesis rests well with the experiments o f B r a a m et al. (1993) which showed that enaprilat, an angiotensin converting e n z y m e inhibitor, did not decrease the concentration of tubular A n g II, as would be expected if A n g II was being produced in the lumen. In contrast, it fits badly with the failure to observe secretory granules in the proximal convoluted tubule or any immunohistochemical staining o f A n g II in any nephron structure besides the intensely staining juxtaglomerular cells (Taugner et al. 1982; Cantin et al. 1984; C.Sernia and M.I. Phillips, unpublished observations). It is our view, based on the totality of the published evidence and our own data, that angiotensinogen is synthesized in proximal convoluted tubule cells and secreted into the lumen where it is used as substrate for A u g II production. In support of this view are data showing renin and angiotensinogen secretion by proximal tubule cells in culture (Yanagawa et al. 1991; Chert et al. 1993). However, it is acknowledged that these in vitro experiments do not unequivocally prove the presence o f these proteins in the lumen o f the proximal convoluted tubule. Furthermore we have not definitively shown that the cytoplasmic stain seen with the light microscope and in electron-dense vesicles is intact angiotensinogen, that it is synthesized in that cell and that it is not of extracellular origin. These are aspects which future investigations need to address.

Acknowledgements. The excellent technical assistance of Michelle Giles and David Kerr is acknowledged with thanks. This work was supported by a grant to Conrad Sernia and an Institute Grant to the Howard Florey Institute from the National Health and Medical Research Council of Australia.

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