Department of Clinical Physiopathology, 1 Endocrinology Unit and 2 Clinic of Urology, University of Florence, 50139 Florence, Italy; and 3 Howard Hughes Medical Institute and Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235
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ABSTRACT |
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The distribution of endothelin-converting enzyme-1 (ECE-1) mRNA and protein was investigated in human kidney excised because of renal tumors. ECE-1 immunoreactivity was detected by immunohistochemistry throughout the different areas of the kidney in the vascular and tubular structures. In the cortex, ECE-1 immunostaining was present in the endothelial surface of arcuate and interlobular arteries and in arterioles. Weak specific immunoreactivity was present over some proximal and distal tubules. Few endothelial glomerular cells contained ECE-1 protein. In the medulla, ECE-1 immunoreactivity was observed in the vasa recta bundles and capillaries. ECE-1 immunostaining was also detected in the outer and inner medullary collecting ducts and thin limbs of Henle's loops. Immunohistochemical detection of the von Willebrand factor on adjacent sections confirmed the endothelial nature of the vascular cells that exhibited ECE-1 immunostaining. The distribution patterns of ECE-1 mRNA, investigated by in situ hybridization, appeared similar to that obtained by immunohistochemistry in the cortical and medullary vasculature and in different portions of the nephron. Northern blot and densitometric analyses demonstrated that ECE-1 mRNA levels were quantitatively similar in both the renal cortex and medulla. These results demonstrate that vascular endothelial and tubular epithelial cells in the cortex and medulla of the human kidney synthesize ECE-1, which, in turn, may play an important role in regulating endothelin production in physiological and pathological conditions.
endothelin; immunohistochemistry; in situ hybridization; Northern blot analysis
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INTRODUCTION |
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THE EXOGENOUS ADMINISTRATION of endothelin-1 (ET-1), a 21-amino acid vasoconstrictor peptide (33), affects both renal hemodynamics and tubular function (for references, see Ref. 26). A growing body of evidence suggests the existence of an intrarenal ET system. In previous observations, ET levels in urine of normal subjects were found to be six-fold higher than in plasma (3). Furthermore, binding sites and gene expression of both types of ET receptors have been localized within mammalian kidney (6, 13), with ETB quantitatively predominating over ETA receptors (13). In addition, gene expression of the precursor of ET-1, prepro-ET-1, has been demonstrated in renal vascular endothelium and nephron segments of different animal species, including the humans (11, 16, 21, 29, 30). Although the detection of ET-1 immunoreactivity and immunostaining in mammalian kidney (12, 31) seems to strengthen the hypothesis that the kidney is a source of mature ET-1, the existence of an intrarenal ET-1 system is critically conditioned by the presence of the processing machinery that generates mature ET-1 from its precursor. In fact, mature ET-1 originates from an intermediate amino acid precursor, big ET-1, through a protheolitic cleavage between Trp21 and Val22 by an ET-1-converting enzyme (ECE-1) (19, 33). Because the biological activity of big ET-1 is negligible (19), ECE-1 is most probably the key enzyme in the biosynthetic pathway of ET, and, therefore, its presence is fundamental for the production of the biologically active form of the peptide within the different structures of the kidney. Biochemical characterization (1, 17, 28) and cDNA cloning (7, 23-25, 32) of ECE-1 in different animal species have revealed that this enzyme is a phosphoramidon-sensitive, membrane-bound, highly glycosilated protein that shares many structural similarities with neutral endopeptidase (EC 24.11) and the Kell blood group protein. ECE-1 cleaves big ET-1 more efficiently than either big ET-2 or big ET-3 (17, 24). Thus the localization of ECE-1 is determinant in targeting the cells capable of generating mature ET-1 and, consequently, in understanding the complex organization of the ET system within the kidney.
Although the presence of ECE-1 mRNA has been univocally demonstrated in mammalian kidney (22, 23, 32), information regarding its intrarenal distribution is limited to the rat (27) and ox (32).
Intrarenally generated ET-1 has been reported to be involved in the regulation of volume homeostasis in humans (15). It also seems to be altered in animal models of acute and chronic renal failure (2, 5) and in chronic progressive nephropathies in humans (20). Consequently, ECE-1 could be a key step in the pharmacological modification of intrarenal ET-1 production.
These considerations prompted us to evaluate the distribution of ECE-1 mRNA and its protein in the human kidney using in situ hybridization and immunohistochemistry.
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MATERIALS AND METHODS |
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Tissues. Kidneys were obtained at
surgery from nine patients affected by localized renal tumors (4 females and 5 males; age range, 42-79 yr). Renal specimens were
cut from the pole opposite to the tumor. Kidney tissue fragments
containing cortex and medulla were subjected to in situ hybridization
or immunohistochemistry, as described below. In other kidney specimens,
the cortex was carefully separated from the medulla, and the tissues
were snap frozen in liquid nitrogen and stored at 70°C until
RNA extraction was carried out as described below.
Immunohistochemistry. Immunohistochemical detection of ECE-1 was performed on four kidneys by the avidin-biotin-peroxidase complex method using a commercially available kit (Vectastain Elite kit, Burlingame, CA). Kidney samples containing cortex and medulla were fixed in 4% paraformaldehyde for 12 h at room temperature and cryoprotected overnight in 30% sucrose at 4°C. Frozen sections (10 µm) were incubated with 0.3% H2O2 in methanol, followed by exposure to normal goat serum. Sections were then layered with a rabbit polyclonal antibody directed against the COOH-terminal 16 amino acids of bovine ECE-1 (4) diluted at 1:4,000-1:5,000. This antibody does not crossreact with the ECE-2 isoenzyme. Biotinylated secondary antibody and the avidin-biotin-peroxidase complex were subsequently applied to the sections; diaminobenzidine tetrahydrochloride was used as peroxidase substrate. All incubations were performed at room temperature, and washing cycles were carried out using phosphate-buffered saline. Finally, sections were counterstained with Harris' hematoxylin and mounted with Permount. To assess the specificity of ECE-1 immunostaining, the primary antibody was preadsorbed with the ECE-1 synthetic 16-amino acid peptide (final concentration, 1 mg/ml) prior to tissue incubation. As additional negative controls, ECE-1 antibody was omitted or replaced by nonimmune rabbit serum. To further clarify the identity of the cells that express ECE-1, immunohistochemistry using an anti-von Willebrand factor (VWf) polyclonal antibody (Dako, Glostrup, Denmark) was performed on consecutive sections using the same technical procedure. The VWf antibody was applied to the sections at dilutions ranging from 1:8,000 to 1:12,000.
In situ hybridization. In situ hybridization was performed on five kidneys, as previously described (21). Briefly, kidney portions containing cortex and medulla were fixed in 4% paraformaldehyde for 12 h at room temperature and cryoprotected overnight in 30% sucrose at 4°C. Frozen sections (10 µm) were thaw mounted onto gelatin-coated slides and dehydrated in increasing ethanol concentrations. Thirty microliters of hybridization solution (40% formamide, 4× standard sodium citrate, 10 mM dithiothreitol, 1× Denhardt's solution, 10% dextran sulfate, 0.1 mg/ml sheared herring sperm DNA, and 1 mg/ml yeast tRNA) containing 8 × 105 counts/min of 35S-labeled human ECE-1 RNA probes, synthesized as described below, were applied to each section and covered with parafilm. Hybridization was carried out at 52°C for 16 h. Removal of the nonspecifically bound probe by ribonuclease digestion, subsequent washing steps, and autoradiography were performed as described elsewhere (21). Sections were subsequently counterstained with hematoxylin-eosin-phloxine and mounted with Permount. An average of eight sections were analyzed for each kidney. Negative controls consisted of hybridization to a sense RNA probe, synthesized as described below.
Human ECE-1 RNA sense and antisense probes were synthesized from a
Hind III-BamH I 1,200-bp fragment of
2,700-bp human ECE-1 cDNA (M. Yanagisawa, personal data) that had been
subcloned in pGEM-4Z plasmid vector (Promega, Madison, WI). The
1,200-bp cDNA was subsequently linearized with Hind III or
BamH I restriction enzymes, followed by
phenol-chloroform extraction and ethanol precipitation. Thereafter,
sense and antisense RNA radiolabeled probes were synthesized using SP6
or T7 RNA polymerases as appropriate (Riboprobe Gemini System, Promega)
in the presence of
[-35S-thio]UTP
(1,300 mCi/mmol, NEN-DuPont, Paris, France). RNA probes were extracted
using phenol-chloroform, ethanol precipitated, and subsequently
subjected to alkaline digestion so as to obtain probes of 150 nucleotides.
To assess the specificity of the low hybridization signal detected on the proximal and distal tubules, the number of silver grains present on these structures were manually quantified in the antisense-hybridized sections and then compared with those detected in the sense-hybridized sections. Ten distal and ten proximal tubules were chosen at random and blindly analyzed in two antisense- and two sense-hybridized sections, taken from each of the five kidney specimens. The number of silver grains per tubule was pooled and averaged for antisense- and sense-hybridized sections. Comparisons were made using Student's t-test for unpaired data considered significant at the level of P < 0.05.
Northern blot analysis. Northern blot analysis was performed on three kidneys as previously described (21).
Briefly, total RNA was obtained from the medulla and the cortex by acid
guanidinium-phenol-chloroform extraction (21). Thirty micrograms of
total RNA, obtained by pooling 10 µg of each sample both from the
cortex and the medulla, were separated by electrophoresis in a 1.2%
agarose-0.7 M formaldehyde gel containing 0.5 µg/ml ethidium bromide.
RNA was subsequently transferred to a nylon membrane (Gene Screen Plus,
NEN-DuPont) and hybridized to a 1,200-bp human ECE-1 cDNA probe
[Hind III-BamH I fragment obtained
from a 2,700-bp cDNA (M. Yanagisawa, personal data)], which is
the same one used for in situ hybridization experiments described above. The cDNA probe was labeled with
[-32P]dCTP (3,000 Ci/mmol; Amersham International, Amersham, UK) by the nick translation
method using a commercial labeling kit (Promega). RNA integrity and
uniformity of loading were evaluated on the gel by ultraviolet
transillumination. Hybridization signals were detected by
autoradiography and quantified by densitometric analysis. ECE-1
autoradiographic signals were normalized to the intensity of the 28S
ribosomal bands.
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RESULTS |
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Immunohistochemistry. ECE-1 immunoreactivity was detected in both the cortex and the medulla of all the kidney specimens evaluated.
In the cortex, ECE-1 immunoreactivity was clearly detected in the endothelial layer of arcuate and interlobular (Fig. 1A) arteries and arterioles. A weak immunoreactivity was also observed in the proximal and distal tubules, although the distribution pattern of ECE-1 immunostaining in these segments of the nephron was not homogeneous. In fact, only some proximal and distal tubules showed specific immunostaining in all the sections examined (Fig. 1A). Few cells exhibited immunoreactivity in the glomeruli (Fig. 1C). A comparison between the distribution patterns of glomerular ECE-1 and VWf immunostaining in consecutive sections suggested that ECE-1 was localized mainly in endothelial cells. However, podocytic epithelium and mesangial cells also occasionally showed specific immunostaining.
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In the medulla, the vasa recta bundles, prevalently descending vessels, and capillaries showed intense immunostaining (Figs. 2, A and C, and 3, A and B). A comparison of ECE-1 and VWf distribution patterns confirmed the endothelial nature of the medullary vascular ECE-1-containing cells (Figs. 2D and 3C). In addition to the vascular network, ECE-1 immunoreactivity appeared to be widely distributed over the tubular structures. Clear and consistent immunostaining was observed in the outer and inner medullary collecting ducts and thin limbs of Henle's loop (Figs. 2A and 3, A and B). Only in some sections was a weak and focal immunoreaction detected in the thick ascending limbs of the Henle's loop (Fig. 2A). ECE-1 was also evident in the pelvic epithelium (Fig. 3B) of two renal specimens that contained this portion of the kidney. Sections in which the primary antibody was omitted or replaced by nonimmune rabbit serum did not show any immunoreactivity (Fig. 3D). Preabsorption of ECE-1 antibody with the synthetic COOH-terminal 16 amino acids of ECE-1 completely abolished the immunostaining (Figs. 1B and 2B).
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In situ hybridization. The distribution patterns of ECE-1 mRNA paralleled those of protein expression, as described above.
In the cortex, ECE-1 mRNA was detected in the endothelial cells of large vessels (Fig. 1D) and arterioles (Fig. 1E). ECE-1 mRNA was also present in the proximal and distal tubules (Fig. 1E). In these tubular structures, the intensity of the hybridization signal was homogeneously low but significantly higher than the one present in those sections hybridized with the sense probe (proximal tubules: antisense, 38.5 ± 6.6, vs. sense, 14.8 ± 3.9, P < 0.0002; distal tubules: antisense, 36.1 ± 6.7, vs. sense, 9.4 ± 2.3, P < 0.0001; means ± SD). Low levels of ECE-1 mRNA were also detected in the capillary loops of the glomeruli.
In the medulla, the vasa recta bundles (Fig. 2E) and capillaries (Fig. 3, F and G) consistently showed dense clusters of silver grains. ECE-1 mRNA was clearly detected, among tubular segments, in the outer and inner medullary collecting ducts and thin limbs of Henle's loops (Figs. 2E and 3, F and G). Specific hybridization signals were detected in the cells of the pelvic epithelium, which was present in one renal specimen (Fig. 3E). Very few scattered silver grains were detectable in the negative control sections hybridized with the sense probe (Fig. 1F).
Northern blot analysis. With the specific Hind III-BamH I 1,200-bp cDNA probe, ECE-1 mRNA was detected as a single band of ~4.6 kb in both the pooled cortex and medulla (Fig. 4A). Total RNA appeared to be not degraded and equally loaded (Fig. 4B).
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The optical density of ECE-1 mRNA autoradiographic signals, normalized to those of 28S ribosomal bands, was only 1.1 higher in the cortex than in the medulla.
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DISCUSSION |
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In the current study, we assessed ECE-1 gene expression in the human kidney and described the intrarenal localization of ECE-1 mRNA and its protein. In situ hybridization and immunohistochemistry revealed that ECE-1 is widely distributed throughout the different areas of the human kidney in vascular endothelial and tubular epithelial cells. Very limited data are available regarding ECE-1 distribution in other species. The limited information available in the rat kidney suggests that there is a moderate level of hybridization signal in endothelial and tubular cells (27). In other studies, immunohistochemical analysis of bovine kidney cortex demonstrated ECE-1 immunoreactivity only in the endothelium of the interlobular arteries, without any specific staining present in glomeruli, proximal, or distal convoluted tubules (32). These discrepancies, related to the tubular localization of ECE-1 immunostaining, may be species dependent or may have been due to technical reasons, including the different antibodies used in the two different studies.
The detection of ECE-1 mRNA in human kidney homogenates confirms previously reported results obtained in the whole organ (23) and in the renal cortex (22). In addition, by quantifying ECE-1 gene expression in the renal cortex and medulla, we were able to demonstrate that similar levels of ECE-1 mRNA transcripts are present in these two areas of the kidney.
The distribution patterns of ECE-1 mRNA and its protein parallel, although not completely, that of prepro-ET-1 mRNA in human kidney (21). The coexpression of ECE-1 and prepro-ET-1 in the same renal structures, together with the presence of ET-1 immunoreactivity in human kidney homogenates (12), is in keeping with the existence of an intrarenal ET system generating mature ET-1, which, in turn, may exert autocrine and/or paracrine actions through the activation of the ETA and ETB receptors.
Indeed, ECE-1 mRNA and its protein were found in the cortical and medullary vasculature of the human kidney, as previously reported for prepro-ET-1 mRNA (21). A comparison between the distribution patterns of ECE-1 and VWf immunostaining confirmed the endothelial nature of these cells. These results, which closely parallel the vascular distribution of ET-1 mRNA and protein in human and animal kidney (11, 16, 31), provide further morphological support to the hypothesis that intrarenally generated ET-1 may regulate renal hemodynamics. This would be in agreement with the effects provoked by the exogenous administration of ET-1 (26).
In addition to the renal vessels, ECE-1 mRNA and protein was demonstrated in the epithelial cells of the collecting ducts where ET-1 synthesis and the presence of ET receptors (6, 13) have been extensively demonstrated in mammalian kidney (9, 21, 29-31). Indeed, in vitro experiments have pointed out a role for ET-1 in the regulation of Na+-K+-adenosinetriphosphatase activity and vasopressin-stimulated osmotic water permeability in these segments of the nephron (14, 34). However, the tubular distribution pattern of ECE-1 appears to be more diffuse than that of ET-1 gene expression. In fact, both ECE-1 mRNA and protein were also localized in the thin limbs of Henle's loop and, to a lesser extent, in the proximal and distal tubules, where previous studies had failed to detect any significant amounts of prepro-ET-1 mRNA in the human and rat kidney (21, 29, 30). Thus it is possible that other tubular segments, besides the collecting ducts, might be potential sources of mature ET-1 synthesis, even if ECE-1 and ET-1 genes are expressed differently at these levels under basal physiological conditions. It is noteworthy that ET-1 immunoreactivity has been demonstrated in the cortical proximal and distal tubules and in the thin limbs of ascending loops of Henle in patients affected by microhematuria (18). Furthermore, the same authors reported that hypoxia results in specific upregulation of prepro-ET-1 mRNA in cultured cells of the proximal tubules (18), suggesting that ET-1 gene expression, at variance with ECE-1, may become relevant only in particular pathological settings. Although ET-1 production seems to be regulated at the transcriptional level (10), the regulation of ECE-1 synthesis is rather unknown.
Alternatively, it might be possible that those tubular cells that do not synthesize ET-1 but express ECE-1 on their cellular surface may generate mature ET-1 from big ET-1 delivered in an endocrine or paracrine fashion. In fact, it is interesting to note that ET-1 has been detected immunohistochemically in the brush border of rat proximal tubules (31).
ECE-1 mRNA and immunoreactivity were limited to a few cells in the glomeruli, the majority of which were endothelial in nature as demonstrated by comparing the distribution of ECE-1 and VWf on consecutive sections. ET-1 mRNA has not been detected in the human glomeruli (21), and immunostaining for ET-1 and big ET-1 has been demonstrated at this level using very thick sections (8). Taken together, these data suggest that glomerular production of mature ET-1 in humans is not high under basal conditions. At variance with these findings, prepro-ET-1 mRNA and ET-1 immunoreactivity have been clearly detected in the rat glomeruli (29-31), suggesting that interspecies differences in ET-1 synthesis might exist in this part of the kidney.
ECE-1 mRNA and protein have been observed in the pelvic epithelium. These data, together with previous finding demonstrating ET-1 immunoreactivity in human pelvic epithelium (18), strongly suggest that this portion of the kidney is a source of ET-1 production. The role of ET-1 in the pelvic epithelium is unclear, but it is possible to speculate that the peptide might contribute to contract the smooth muscle cells of the calyces and pelvis wall to propel the urine throughout the ureter to the bladder.
Although changes in ET-1 gene expression have been documented in experimental models of renal diseases (2, 5), very little information is available regarding urinary ET-1 levels in human renal diseases (20). Furthermore, the role of ECE-1 in human renal pathologies must still be clarified. Further studies, which can be performed successfully by applying the techniques developed in the present study, will be necessary to address this issue.
In summary, we established that ECE-1 mRNA and its protein are widely distributed in the vascular and tubular structures of the human kidney. Thus the organization of the ET system in this organ can be better depicted. ECE-1 could represent a key step in regulating the paracrine and/or autocrine actions of ET-1 within the kidney in physiological as well as in pathological conditions.
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ACKNOWLEDGEMENTS |
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We thank the staff at the Urology Clinic, University of Florence, for their help in collecting renal samples.
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FOOTNOTES |
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These studies were supported by a grant from the Ministero dell' Università e della Ricerca Scientifica e Tecnologica.
The results of the present study were reported in preliminary form at the Giornate Endocrinologiche Pisane, Pisa, Italy, 27-28 June, 1996, and have appeared in abstract form (J. Endocrinol. Invest. 19, Suppl. 5: 28, 1996).
Address for reprint requests: C. Pupilli, Dipartimento di Fisiopatologia Clinica, Unita' di Endocrinologia, Viale Pieraccini 6, 50139 Florence, Italy.
Received 26 September 1996; accepted in final form 7 July 1997.
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