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Department of Medicine (N.A., A.A., G.N.H., D.G.) and Physiology (H.S.L., M.Y.K., G.N.H., J.H.W., D.G.), and Anatomy and Cell Biology (H.W.), McGill University, and Calcium Research Laboratory (N.A., A.A., G.N.H., D.G.), Royal Victoria Hospital, Montreal, Quebec H3G 1Y6, Canada; and the Department of Oral Anatomy (N.A., H.O.), Niigata University School of Dentistry, 5274, 2-Bancho Gakkoucho-Dori, Niigata, 951 Japan
Address all correspondence and requests for reprints to: J. H. White, Department of Physiology, McIntyre Medical Sciences Building, Room 1109, McGill University, 3655 Drummond Street, Quebec H3G 1Y6, Canada. E-mail: jwhite{at}physio.mcgill.ca
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (1). PTH enhances calcium
reabsorption in the renal distal convoluted tubule and inhibits
phosphate and bicarbonate uptake in the proximal convoluted tubule.
PTH-related peptide (PTHrP), originally described as the major mediator
of hypercalcemia associated with a variety of malignancies (2), can
mimic many of the effects of PTH when overproduced by cancers. Under
physiological conditions, PTHrP is thought to act in a
paracrine/autocrine mode, in contrast to PTH. Moreover, whereas PTH
expression is restricted to the parathyroids, PTHrP is widely expressed
and functions to modulate normal cellular growth and differentiation
(3, 4, 5, 6). The most striking evidence for a physiological role for PTHrP
has come from gene ablation experiments in mice that have shown that
PTHrP is essential for normal endochondral ossification (7, 8, 9). In the
adult, PTHrP also has been shown to act as a relaxant of smooth muscle,
including vascular smooth muscle (10). Unlike the C-terminal portions of PTH and PTHrP, the N-terminal 34 amino acids of the two hormones are partially conserved and have been postulated to form similar helix-turn-helix structures (11). The spectrum of actions common to PTH and PTHrP emanates from the capacity of the conserved N-terminal region of each hormone to interact with the same guanylyl-nucleotide binding (G) protein-coupled receptor. The complementary DNA (cDNA) and gene encoding the PTH/PTHrP receptor (PTHR) have been cloned, and like PTHrP, the PTHR is widely expressed (12).
Recently, a second PTH receptor (PTH2), with only 20% homology to PTHR, has been cloned. PTH2 is mainly localized to brain and, to some extent, in pancreas, testes, and placenta but not in kidney (13). This receptor seems to bind PTH with much higher affinity than PTHrP.
The mouse gene encoding the PTHR is composed of at least 17 exons, and its expression is regulated by at least two promoters (14, 15). These promoters give rise to two transcripts differing in their 5' untranslated sequences but not their coding regions. Transcripts expressed from the upstream promoter (P1) contain sequences derived from two 5' untranslated region exons U1 and U2. Expression of P1 is largely restricted to kidney although weak expression is detected in liver (15). The downstream mouse promoter (iP2) is very (G+C)-rich and is widely expressed (15). P2-specific transcripts contain 5' untranslated sequences derived from exon U3. RNase protection assays have suggested that P1 activity accounts for 80–90% of PTHR transcripts detected in the mouse kidney. Recently, the 5' end of the highly conserved rat PTHR gene has been found to have a very similar exonic structure and pattern of transcript expression (16). cDNAs have been isolated from human kidney cDNA libraries that contain sequences 74% similar to mouse exons U1 and U2, suggesting that the structure of the 5' end of the PTHR gene also is conserved in humans (17).
The combined length of exons U1 and U2 is very similar to that of U3 (15), which accounts for the detection in most tissues of a single messenger RNA (mRNA) of 2.3 kilobase pairs (kb) (18, 19). However, larger and smaller mRNA species have been detected with PTHR cDNA probes in rat kidney, liver, brain, skin, and testes (18, 19). In addition, a receptor with different intracellular signaling properties has been detected in normal keratinocytes and malignant squamous cells (20). Whether these species represent distinct gene products or alternatively spliced forms of the PTHR remains to be determined.
The PTHR is most highly expressed in the kidney, where its action is essential for ion homeostasis. We are interested in analyzing the regulation of expression of the receptor in the kidney. Although our previous studies suggested renal PTHR expression was driven largely by the P1 promoter, gene transfer experiments presented below suggested that P2 also is active in renal epithelial cell lines. We have therefore examined the localization of PTHR protein and P1- and P2-derived mRNAs in the different tissues in the kidney by immunocytochemistry and in situ hybridization using transcript-specific probes. Our results demonstrate that both P1 and P2 are expressed in epithelial cells of the nephron and provide evidence for strong PTHR gene expression driven exclusively by P1 in renal vasculature.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Northern analysis
Northern hybridizations were performed essentially as
described (21). Briefly, 20–30 µg of mouse kidney total RNA was
resolved on 1% agarose formaldehyde gel and blotted onto nylon
membrane (Amersham, Oakville, Ontario, Canada). Prehybridization and
hybridization were carried out in 6 x SSC, 5 x Denhardt’s,
1% SDS, 100 µg/ml salmon sperm DNA, and 10% dextran sulfate at 65
C. 32P-labeled probes were synthesized by random primed
labeling of DNA fragments excised from plasmids pU1/U2
(XbaI/XhoI), pU3/170
(ApaI/XbaI), and KS-rPTHrec4-1
(AccI/HindIII; 171 bp of coding sequence encompassing
transmembrane region a.a. 387–427).
Cell culture and lipofections
Renal epithelial cell lines Madin-Darby canine kidney, mouse
distal convoluted tubule (a generous gift of Dr. P. Friedman,
Dartmouth, NH), and opossum kidney OK cells were cultured in DMEM/F12
media (GIBCO, Burlington, Ontario, Canada) supplemented with 50 U/ml
each of penicillin and streptomycin and 10% FBS (GIBCO). Cells were
transfected by lipofection using Lipofectin (GIBCO), according to the
manufacturer’s instructions, with 1 µg of ß-galactosidase
expression vector p610AZ (15) and 2 µg of PTHR promoter recombinants.
Transfection efficiencies for a given cell type varied by ± 20%.
Cells were harvested in 300 µl Reporter Lysis Buffer (Promega,
Madison, WI) and 30 µl used for ß-galactosidase assays (to
normalize for transfection efficiency) and for luciferase assays
(Promega).
Immunohistochemistry for the PTHR
For the PTH receptor immunohistochemistry, 7- to 8-week-old male
ddY mice (Japan SLC Inc., Hamamatsu, Japan) were anesthetized with
nembutal and perfused through the left ventricle with 4%
paraformaldehyde diluted in 0.1 M phosphate buffer (pH
7.4). The kidneys were extracted and immersed in the same fixative for
6 h at 4 C. Some specimens were dehydrated with an increasing
concentration of ethanol before paraffin imbedding, and the others were
immersed in a series of ascending concentrations from 5–25% sucrose
in PBS. Paraffin or frozen cryostat sections of approximately 8 µm
thickness were collected on poly-L-lysine-coated glass slides.
Rabbit polyclonal antisera were raised against epitopes in the
N-terminal extracellular domain of the rat PTHR. Antiserum no.
IK-XXI-89 was directed against amino acids 85–105
(GKFYPESKENKDVPTGSRRRG), which is 100% conserved in the mouse (14).
This antibody recognizes the PTHR specifically as judged by Western
analysis (22) and has been used previously to detect PTHR expression in
rat tibiae (22). The dewaxed paraffin sections were pretreated with
0.3% H2O2 in PBS for 10 min at room
temperature to inhibit endogenous peroxidase activity and subsequently
treated with 1% BSA in PBS (BSA-PBS) for 30 min at room temperature.
They were incubated with no. IK-XXI-89 at a dilution of 1:100 in 1%
BSA-PBS overnight at 4 C. After rinsing with PBS, the sections were
incubated with horseradish peroxidase-conjugated goat f(ab')2
fragment-antirabbit IgG (Calbiochem, San Diego, CA) at a dilution of
1:100 with 1% BSA-PBS for 60 min at room temperature. Visualization
for the immunohistochemistry was performed using diaminobenzidine as a
substrate. The specificity of signals obtained by immunohistochemistry
were confirmed using nonimmune serum, which gave no signal on paraffin
sections of mouse kidney cortex or medulla. As a further index of
specificity, immunocytochemistry was performed on Chinese hamster ovary
(CHO) cells transiently transfected with PTHR cDNA by the calcium
phosphate technique (Fig. 1
). Untransfected CHO cells
served as a negative control. A specific signal was seen only in cells
expressing the PTHR using anti-PTHR antiserum. No specific signal was
observed in immunocytochemical experiments performed with transfected
cells or tissues in the absence of anti-PTHR antiserum.
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In situ hybridization
Sprague Dawley rats were anesthetized with nembutal, and kidneys
were extracted immediately and immersed in liquid nitrogen. Frozen
sections of the kidneys were collected on poly-L-lysine-coated glass
slides (Polyscience Inc.). Sections were subsequently treated with 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for
15–20 min. and then with proteinase K (10 µg/ml) in 10
mM Tris-HCl (pH 8.0) at 37 C for 3–5 min. Acetylation of
sections was performed by incubation for 10 min. with 0.25% acetic
anhydride in 0.1 M triethanolamine (pH 8.0). cRNA probes
were prepared with digoxigenin-labeled UTP (Boehringer Mannhein
Biochemical, Mannheim, Germany). Sense and antisense cRNA probes for
the rat PTHR were obtained from cloned cDNA plasmid KS-rPTHrec4-1 using
T7 and SP6 RNA polymerases, respectively.
For hybridizations, hybridization buffer [50% formamide, 10 mM Tris-HCl (pH 7.6), 100 µg transfer RNA, 1 x Denhardt’s solution, 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, and 1 mM EDTA] was preheated for 10 min at 90 C. Each cRNA probe was adjusted to a concentration of 0.1–1.0 µg/ml. Hybridization was performed overnight at 50 C, and then sections were washed with 50% formamide in 2 x SSC at 55 C for 30 min and treated at 37 C with a solution of 10 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 1 mM EDTA. Nonspecific binding of probes was reduced by RNase A treatment [20 µg/ml in 10 mM Tris (pH 7.5), 1 mM EDTA, 500 mM NaCl] at 37 C for 30 min. For immunodetection of digoxigenin-labeled probes, sections were preincubated with 2% blocking agent and incubated for 45–60 min with alkaline phosphatase-conjugated sheep antidigoxigenin antibody at a dilution of 1:1000. Visualization was performed using nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolylphosphate. Sense cRNA probes were employed to verify the specificity of hybridization conditions.
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Results |
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). In addition, immunoreactivity was
detected on the luminal surfaces of some tubules (Fig. 2A). In
contrast, only weak immunoreactivity was detected under the same
conditions in glomeruli. No specific signal was observed using
nonimmune serum (Fig. 2B). The distribution of PTHR protein in the
cortex was further investigated by immunoelectron microscopy. PTHR
protein was detected on basolateral membranes and, to a lesser extent,
on the luminal surface of epithelial cells of proximal convoluted
tubule cells (Fig. 3), confirming the results obtained
by light microscopy. Immunopositivity also was seen in vesicles
adjacent to the luminal membrane (Fig. 3B). Strikingly, we also
observed high levels of expression of PTHR protein on the thin walls of
endothelial cells of the cortical capillaries (Fig. 3, A, C, and D).
This immunopositivity was severalfold more intense than that observed
on basolateral membranes of adjacent epithelial cells. We also detected
expression of PTHR protein on the surface of glomerular podocytes (Fig. 4), consistent with our previous findings (23), whereas
no PTHR expression was detected in vascular endothelial cells in
glomeruli.
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).
These results are consistent with previous radioautographic detection
of specific binding of PTH to this site of the nephron (23). Having
examined the distribution of expression of PTHR protein in renal
tissues, we were interested next in determining the distribution of the
different species of PTHR mRNA expressed in the kidney
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). The coding region probe detects a predominant band
of approximately 2.3 kb (Fig. 6B, lane C) (18, 19). P1- and P2-specific
probes recognize distinct 5' untranslated regions but detect major
transcripts indistinguishable in size from those identified with the
coding region probe (Fig. 6B, lanes P1 and P2). These results are
consistent with the large majority of transcripts derived from P1 and
P2, differing in their 5' untranslated regions but not in their coding
sequence (14, 15), although we cannot rule out the possibility of
alternative splicing of the coding region of the gene.
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), suggesting that the P1 and P2
promoters account for the large majority of PTHR gene transcription in
the kidney. In contrast to the expression pattern in kidney, Northern
analysis of PTHR transcripts in heart (Fig. 6C) did not detect any
P1-specific transcripts, whereas the P2-specific probe detected a
2.3-kb band of similar intensity to that seen with the coding sequence
probe. These results serve as an important control by inferring that
the relatively weak expression of P2-specific transcripts seen in
kidney was not caused by inefficient hybridization of the probe.
The relatively high levels of renal expression of the P1 promoter are
striking, given the results of a series of gene transfer experiments
performed in the renal epithelial cell lines OK, mouse distal
convoluted tubule, and Madin-Darby canine kidney using P1- and
P2-promoter-luciferase constructs (Table 1). These
experiments suggested that the P2 promoter showed comparable or greater
activity than P1 sequences. Similar results were obtained in
transiently transfected OK cells with P1 and P2 promoter recombinants
containing 5.7 and 6 kb of flanking sequence, respectively (data not
shown). The apparent discrepancy between the relative expression of P1-
and P2-specific transcripts in total kidney detected by Northern
analysis and the relative activities of our reporter constructs in
epithelial cells indicated that it was important to determine the
expression patterns of the two promoters in different renal tissues
before undertaking comprehensive promoter analyses.
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). Intense hybridization signals
specific for the PTHR coding region were observed in epithelial cells
of cortical tubules (Fig. 7, A and D). Consistent with
observation of PTHR protein in glomeruli (Fig. 4), coding
sequence-specific hybridization also was observed in some, but not all,
cells in glomeruli (Fig. 7A). A very similar pattern of expression was
detected with P1-specific sequences. Extensive hybridization was
detected in cortical epithelial cells (Fig. 7, B and E) along with
focal hybridization in glomeruli (Fig. 7B). In addition, we observed
intense hybridization in capillary endothelial cells located adjacent
to tubules with both coding region and P1 probes (Fig. 7, D and E). No
specific signals were observed with sense probes corresponding to
coding region-specific and P1-specific sequences (Fig. 7, G and H).
These results are consistent with the abundance of PTHR protein
observed in endothelial cells lining cortical capillaries (Fig. 4).
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, C, F, and I). No (or only weak) expression of
P2-specific transcripts was detected with an antisense probe in
glomeruli (Fig. 7C), and no P2-specific expression was seen in vascular
endothelial cells (Fig. 7, C and F; and data not shown). This indicates
that expression of the PTHR gene in glomeruli and in vascular
endothelial cells is controlled exclusively by the kidney-specific P1
promoter.
Expression of PTHR transcripts in the medulla
We also analyzed expression of PTHR transcripts in longitudinal
and cross-sections taken from medulla. PTHR transcripts were detected
using a coding region probe in collecting ducts in longitudinal and
cross-sections of the medulla (Fig. 8, A and C). A
similar expression pattern was observed in the medulla with a
P1-specific probe (Fig. 8, B and D). Further evidence for PTHR
transcript expression in the renal medulla was seen at lower
magnification (Fig. 9). Coding
sequence and P1-specific expression was clearly evident (Fig. 9, A and
C), whereas P2-specific expression was virtually undetectable (Fig. 9E). These results are consistent with immunohistochemical analyses
presented in Fig. 5.
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, A–D). This
signal persisted even after washing sections under high stringency (not
shown). In contrast, no specific signal was observed with a P2-specific
probe (Fig. 9, E and F), indicating that P2 expression is very low or
nonexistent in these cells. These results provide evidence for high
levels of P1-specific PTHR mRNA expression in renal vascular smooth
muscle. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PTHR protein was detected on both the basolateral and luminal surfaces of cortical epithelial cells by immunoelectron microscopy. The dual delivery of circulating, bioactive PTH to the nephron by glomerular filtration and by peritubular uptake, which might result in PTH action on both brush border and basolateral membrane, has been previously reported (24). Our demonstration of the bipolar distribution of PTHR protein in proximal tubule cells, therefore, provides a structural basis for such interactions, and for studies demonstrating PTH binding, and PTH-stimulated adenylate cyclase on basolateral and brush border surfaces (23, 25). It is possible that the receptors we identified on the apical surface are bound by PTHrP that has translocated from the cytoplasm to the subapical region after the induction of ischemia (26).
PTH exerts pleiotropic effects in the renal tubule. Although the
precise distribution of the different functions of PTH may vary from
species to species, several approaches have localized PTH-mediated
inhibition of phosphate (27) and bicarbonate reabsorption (28) and
stimulation of 25 hydroxyvitamin D 1 hydroxylase activity to the
proximal tubule. PTH stimulation of calcium reabsorption has been
localized to the ascending limb of Henle’s loop (29) and to portions
of the distal tubule (30). Our studies, demonstrating the presence of
PTHR mRNA and protein along the nephron, correlate well with results
obtained with physiological approaches (31).
PTH binding in vivo (23) and in vitro (32) and adenylate cyclase stimulation in vitro (33) have been reported in glomeruli. In addition, PTH has been shown to influence glomerular filtration in vivo (34). Our immunoelectron microscopic analyses have detected PTHR protein on the surface of podocytes, and our in situ hybridization studies demonstrated expression of PTHR transcripts in selected cells in glomeruli. Of special importance is the finding that PTHR expression in these cells was derived from kidney-specific promoter P1 but not from ubiquitous promoter P2.
Immunoelectron microscopic analyses detected high levels of PTHR protein on endothelial cells of the peritubular vasculature. In situ hybridization studies with promoter-specific probes showed that this expression is controlled exclusively by the kidney-specific P1 promoter of the PTHR gene. Moreover, P1, but not P2, is strongly expressed in vascular smooth muscle of the kidney. These striking observations indicate that high levels of PTHR are expressed in renal vascular tissue, either in endothelial or smooth muscle cells. Both PTH and PTHrP exert hypotensive effects mediated by their N-terminal domains when they are infused in vivo, and both are believed to directly interact with blood vessels (35). Abundant PTHrP expression has been reported in rat aortic smooth muscle cells, which also respond to the peptide, consistent with an autocrine/paracrine effect of PTHrP on the vasculature (36). PTHrP also is known to be expressed in kidney (26) and may therefore play an autoregulatory role as a renal autocrine/paracrine factor. Thus, in the kidney, both PTH and PTHrP can increase regional blood flow and/or decrease vascular resistance (35) and can relax the renal artery in the preconstricted rat kidney (37). Both hormones bind to vascular smooth muscle of rabbit renal microvessels (38). Furthermore, both endothelium-dependent nitric oxide-mediated vasodilatation (39) and endothelium-independent cAMP-stimulated vasodilatation (40) have been implicated in the mechanism of vascular action of PTH and PTHrP.
Our present studies documenting the presence of the PTHR in renal endothelial cells and in vascular smooth muscle therefore provide a molecular basis for the action of PTHrP and PTH in the renal vasculature through stimulation of endothelium-related and endothelium-independent events. It is also noteworthy that the microvasculature has not been implicated in control of vascular tension. This raises the possibility that the high levels of PTHR expressed in the endothelium of peritubular capillaries may exert some of the physiological effects of PTH on adjacent epithelial cells of the nephron through endothelium-based signaling events.
Several studies have shown that the kidney is the major site of
expression of PTHR mRNA (15, 18). Our previous RNase protection
experiments indicated that activity of the PTHR P1 promoter is largely
restricted to kidney (15), and moreover, that P1 expression is
responsible for at least 90% of renal PTHR transcripts (Fig. 6).
Nevertheless, P1 and P2 promoter sequences stimulated similar levels of
reporter gene expression in gene transfer experiments in several renal
epithelial cell lines (Table 1). This apparent discrepancy was resolved
by our finding that P1-, but not P2-, specific transcripts were
detected in renal vascular endothelial and smooth muscle tissue. It is
noteworthy that P1 is only weakly active in liver, and its activity is
undetectable in heart (two organs with abundant vascular tissue),
whereas P2 is active in both tissues (15). Taken together, the above
results suggest that expression of PTHR in renal vasculature is
regulated differently from its expression in the vasculature of other
tissues. Therefore, further studies will be necessary to determine the
precise control of P1 in renal vs. extrarenal vascular
tissues and to examine the different regulatory signals modulating P1
and P2 expression in the kidney.
In summary, our immunohistochemical and in situ hybridization studies have documented broad expression of the PTHR in the kidney and emphasize the abundant expression of the receptor in vascular tissues, particularly in peritubular capillary endothelial cells. Promoter-specific in situ hybridization experiments showed that both P1- and P2-specific PTHR transcripts are present in epithelial cells of the nephron, whereas strong P1-specific expression was found in the renal vasculature. Our results are consistent with the PTHR being the predominant receptor for PTH in the kidney. Moreover, they underline the importance of PTH signaling in the renal vasculature through stimulation of events mediated by both endothelial and nonendothelial cells.
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Acknowledgments |
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Footnotes |
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2 N. Amizuka was the recipient of a Research Fellowship from the Royal
Victoria Hospital and currently is the recipient of a fellowship from
the Naito Foundation. G. N. Hendy is a Medical Research Council of
Canada Scientist. J. H. White is a chercheur-boursier of the Fonds de
Recherche en Santé du Québec.
Received May 13, 1996.
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