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Institute of Pharmacology and Toxicology (R.P., M.K.) and Department of Internal Medicine, Experimental Nephrology, Westfaelische Wilhelms-Universitaet Muenster, Muenster 48129, Germany; Institute of Cell Biology (E.E.), ETH Hoenggerberg, Zuerich 8093, Switzerland; and Division of Pediatric Endocrinology (L.A.S.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2579
Address all correspondence and requests for reprints to: Dr. Michaela Kuhn, Department of Pharmacology and Toxicology, Westfaelische Wilhelms-Universitaet Muenster, Domagkstrasse 12, D-48129 Muenster, Germany. E-mail: mkuhn{at}uni-muenster.de
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Abstract |
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Renal uroguanylin messenger RNA expression was higher than guanylin and had a different distribution pattern, with highest levels in the proximal tubules, whereas guanylin was mainly expressed in the collecting ducts. Uroguanylin expression was significantly lower in GC-C-/- mice than in GC-A-/- and wild-types, suggesting that absence of a receptor was able to down-regulate ligand expression. Salt-loading (1% NaCl in drinking water) increased uroguanylin-mRNA expression by >1.8-fold but had no effect on guanylin expression. Uroguanylin but not guanylin transcripts were detected in M-1 cells and increased in response to hypertonic media (+NaCl or mannitol). Our results indicate that high-salt intake increases uroguanylin but not guanylin expression in the mouse kidney. The synthesis of these peptides by tubular epithelium may contribute to the local control of renal function and its adaptation to dietary salt.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the intestine, guanylin and uroguanylin activate CFTR-dependent chloride and bicarbonate secretion, which ultimately drives the paracellular movement of Na+ into the lumen (3, 4, 5, 6). These effects are mediated by a receptor guanylyl cyclase, GC-C, located in the apical, brush-border membrane of the intestinal epithelium. Recent work by Shailubhai et al. (7) has shown that they may also modulate epithelial cell proliferation and apoptosis through GC-C. Besides these local actions in the gut, both peptides, particularly uroguanylin, are postulated to function as endocrine intestinal natriuretic hormones because: 1) both circulate in the bloodstream (8, 9); 2) very high concentrations of uroguanylin are excreted in urine (10); 3) exogenous guanylin and uroguanylin initiate a diuretic, natriuretic and kaliuretic response in rats and mice, both in vivo and in vitro, in the isolated perfused kidney model (11, 12, 13); and 4) uroguanylin is substantially more potent than guanylin in eliciting these renal effects. Thus, these peptides possibly complement the renal effects of the cardiac natriuretic peptides, ANP and BNP.
Somewhat surprisingly, recent work from several laboratories has shown that uroguanylin and guanylin are also detected in the kidney itself (14, 15, 16). Thus, uroguanylin and/or guanylin and their receptors apparently form not only an intestinal system regulating intestinal and distal kidney function, but also a local, intrarenal paracrine, and/or autocrine system modulating kidney function directly. However, very little is known about the precise distribution and regulation of these peptides in the kidney. It is unclear whether their renal expression is regulated by physiological conditions such as increased dietary salt and extracellular volume expansion. It is also not known whether their expression is altered by pathophysiological conditions, such as a decreased action of other endogenous natriuretic systems, i.e. the ANP-guanylyl cyclase A (GC-A) system, or decreased expression of their putative receptor, GC-C.
Previous work in vivo/in vitro has shown that intestinal guanylin and uroguanylin messenger RNA (mRNA) levels are modulated by oral salt and also by neuronal transmitters such as muscarinic agonists and nitric oxide/NO (17, 18, 19). But it is not known whether these conditions are associated with altered renal expression. The major aim of our study was to examine the distribution of uroguanylin and guanylin mRNA in specific segments of the mouse nephron and to determine whether changes in dietary salt or water intake would modulate the renal expression of these peptides. In addition we evaluated how this system is influenced by a chronic deletion of the ANP/GC-A system or by a permanent removal of a putative receptor, GC-C. Disruption of the ANP or GC-C systems was achieved by taking advantage of respective gene knockout mouse models (20, 21). Finally, because in vivo studies on the regulation of renal hormone expression are complicated because of multiple neurohormonal alterations and other physiological factors, we also evaluated uroguanylin expression in M-1 cells, a cell line derived from murine cortical collecting ducts (22).
We show that uroguanylin and guanylin differ not only in their overall expression level, but also in their distribution along the mouse nephron. Uroguanylin expression was highest in the proximal tubules and guanylin was mainly located to the collecting ducts. Renal uroguanylin levels were not altered in a model of ANP-receptor-deficiency and chronic hypertension (GC-A -/- mice), but were significantly reduced in GC-C -/- mice. A 3-day-exposure of mice to high-salt diet (1% NaCl in drinking water) or a 24-h exposure of M-1 cells to hypertonic media markedly increased renal uroguanylin mRNA-expression. In contrast, renal guanylin levels were not affected by salt-load. Together, these results strongly suggest that these peptides play a key role in the local control of tubular electrolyte and water transport and renal adaptation to high salt ingestion.
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Materials and Methods |
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Renal tubule isolation
Individual nephron segments were isolated from collagenase-
digested wild-type mouse kidneys using techniques described in
detail previously (26). Small (approximately 2–3
mm3 pieces) of kidney cortex were incubated in 2
ml of Eagle’s MEM containing 0.5 mg/ml of type 2 collagenase, 5
mM glycine, 50 U/ml desoxyribonuclease, and 48 µg/ml of
soybean trypsin inhibitor (all Sigma, Deisenhofen,
Germany; except the collagenase, which was from Worthington Biochemical Corp., NJ) for 40–50 min at 37 C. Digested tubule
suspensions were rinsed in ice-cold collagenase-free MEM. Nephron
segments were sorted under stereomicroscopic magnification in Petri
dishes containing MEM with 0.05% BSA at 4 C. Glomeruli, proximal
tubules, thick ascending limbs of Henle’s loop and collecting ducts
were collected separately. About 30 units of each nephron segment were
transferred to RNA-extraction buffer (RNeasy Kit, QIAGEN,
Hilden, Germany) and stored at -80 C before RNA extraction and RT-PCR
analysis.
Northern blot and RT-PCR analysis of mouse tissues
Total RNA was isolated by RNeasy Kit (QIAGEN,
Hilden, Germany). For Northern blot analysis 20 µg total RNA were
used. Specific hybridization probes were prepared by complementary DNA
(cDNA) synthesis and PCR amplification using RNA from mouse intestine.
For cDNA synthesis, 1 µg of total RNA was reverse transcribed with
random primers (Advantage, CLONTECH Laboratories, Inc.,
Palo Alto, CA). The primers used for specific PCR amplification were as
follows: For uroguanylin: forward, 5'-GAACCCAGAGGTGTGAGC; reverse,
5'-CTGTGGGGAAAGTGTCC (PCR product 401 bp); For guanylin: forward,
5'-TTGGCTGTCCTGGTAGAAG; reverse, 5'-TGTGGCAGGGCAATAGATG (PCR product
414 bp). For cGMP-dependent protein kinase type II (cGKII): forward,
5'-ACGGGGAGAAACTATCAACAGG; reverse, 5'-GTTTCTCGATCTATCACGAGGC (PCR
product 582 bp); For GC-A: forward, 5'-TATCATCCCAGCATCCTTCCATG;
reverse, 5'-TCGTGTCAACTCAATGCGTTTCC (PCR product 693 bp); For mouse
GC-C, the following specific primers were designed based on sequence
information provided by Dr. J. M. Lopez (HHMI, Dallas/Texas;
personal communication (21): forward,
5'-GACTGGACATAGTGCGAAAGC; reverse 1,5'-GTGGACTGTTCAGACGGCAAC;
reverse 2, 5'-CGTCTCAGTACATCCTTGAAG. These primers were used for
conventional and nested PCR, yielding to PCR products of 723 bp and 502
bp, both containing the extracellular region of GC-C. All PCR products
were verified once by sequence analysis. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used for normalization.
Quantitative real-time RT-PCR
A quantitative analysis of specific mRNA expression was
performed by RT-PCR using the LightCycler Detection System
(Roche, Mannheim, Germany). The system uses two
fluorogenic probes to generate sequence-specific fluorescent
signals during PCR. The probes are oligonucleotides with donor and
acceptor dyes attached, designed from the mRNA sequence to hybridize
with a region between the forward and reverse PCR primers (see above).
When the probes form part of a replication complex the distance between
them is just 1 bp, resulting in a fluorescent signal. The following
probes were used (all designed and synthesized by Tib Molbiol, Berlin,
Germany): for uroguanylin: detection probe, 5'-LC
Red640-ACGGCAGCCCACAGTTGGC-p, anchor probe:
5'-CGCAGGAGCAGCAGCAGGAC-fluorescein; for guanylin: detection probe,
5'-LC Red640-CTCTCAGCCCATGTGGAAGCC-p, anchor probe,
5'-TGCTAGAGTGACATCGCTTGCCTT-fluorescein; for GAPDH, detection probe LC
Red640-AGAGGCCCTATCCCAACTCGGC-p, anchor probe
CCCCAGCAAGGACACTGAGCAA-fluorescein. For PCR, 40 cycles of the following
were run: denaturation at 95 C for 1 sec, annealing at 61 C for 15 sec,
and extension at 72 C for 18 sec. Quantitative PCR analysis was
performed using the LightCycler Software (Roche
Diagnostics, Mannheim, Germany) using a real-time fluorogenic detection
system for a kinetic approach (27). The quantitative data
were calculated from the kinetic curve of the PCR by interpolation with
a standard curve generated using known amounts of the target DNA.
Uroguanylin and guanylin transcripts were normalized to GAPDH
(27).
Culture of murine cortical collecting duct (M-1) cells
M-1 cells, derived from dissected cortical collecting ducts of
mice transgenic for the early region of simian virus 40, have been
shown to retain many characteristics of cortical collecting ducts
(22). The M-1 cells used in our study were generously
supplied by Dr. Jens Leipziger (Department of Physiology, University of
Freiburg, Germany). For experimentation, cells of passages 37–38 were
seeded on semipermeable fibronectin-coated polyester membranes
(Transwell, Corning Costar, Bodenheim, Germany). Culture
medium was a 1:1 mixture of DMEM and Ham’s F-12 (Life Technologies, Inc., Eggenstein, Germany), supplemented with 5%
FCS and 50 U/ml penicillin/50 µg/ml streptomycin. After 5 days, serum
was withdrawn from the culture media. Experiments were performed
24 h later.
Effect of hypertonicity and steroid hormones on uroguanylin mRNA
expression in M-1 cells
Cells were incubated in serum-free medium made hypertonic by the
addition of either 100 mM mannitol (n = 8) or an
additional 25 or 50 mM NaCl (basal NaCl concentration in
culture medium was 119 mM) (n = 9). Parallel
experiments were performed to assess whether the steroid hormone
dexamethasone (0.1 µM, n = 9) modifies uroguanylin
mRNA levels. Agents (all Sigma, Deisenhofen, Germany) were
always added to the apical as well as basolateral side of cultured
epithelia. Untreated cells served as controls (n = 10). After 24-h
cells were harvested in lysis buffer for RNA extraction and
quantitative RT-PCR.
For immunocytochemistry M-1 cells were seeded on fibronectin-coated plastic dishes and treated exactly the same way.
Assay of cGMP in M-1 cells
M-1 cells cultured in 12-well plastic dishes were pretreated
with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX,
1 mM) for 15 min and then treated with ANP, B-, C-type
natriuretic peptide (BNP, CNP), or sodium nitroprusside (SNP) for 5
min, or with guanylin or uroguanylin for 30 min. The incubation medium
was aspirated and intracellular cGMP content was measured by RIA
(8). Rat ANP and human CNP were from Novabiochem (Bad
Soden, Germany), mouse BNP was from American Peptide Co.
(Sunnyvale, CA), IBMX and SNP were from Sigma. Guanylin
and uroguanylin were synthesized by Dr. Knut Adermann, IPF (Hannover,
Germany) (28)
Immunocytochemistry and confocal microscopy
After fixation of cultured M-1 cells in 4% PFA for 10 min and
permeabilization with 0.2% Triton X-100 for 10 min double
immunofluorescent staining was performed as described previously
(29). Primary polyclonal antibodies against uroguanylin
(30) were used together with secondary FITC-conjugated
antirabbit IGs (Cappel, ICN Biomedicals, Eschwege,
Germany) and rhodamine-phalloidin (Molecular Probes, Inc.,
Leiden, The Netherlands) to visualize F-actin. To ensure the
specificity of the fluorescent signal, preabsorption experiments were
included as well, incubating the anti-uroguanylin antibodies at 4 C
over night with the peptide that was used for immunization
(30). Single confocal sections were recorded in a
Leica Corp. confocal microscope (Leica Corp.,
Glattbrugg, Switzerland).
Immunoblot analysis
To compare the expression of GC-C in the rat kidney and
intestine, two well characterized affinity purified antibodies against
the C-terminal peptide of rat GC-C (CNNSDHDSTYF) and against a peptide
from the extracellular domain (CRRSEQFQEILMGRNRKSN) were used
(30, 31). Both sequences are unique to GC-C. Membrane
samples and immunoblots were prepared as previously described
(30, 31). The proteins were solubilized in Laemmli buffer,
resolved by 7% PAGE, and transferred to nitrocellulose. Equal amounts
of membrane protein (50 µg) were loaded per lane. We used primary
antiserum at a concentration of 1 µl/ml of buffer. Preliminary
experiments showed that the immunoreactive bands were specific in that
the reactivity was blocked by preincubating the antibody solution with
the purified peptide.
Statistics
Results are expressed as the mean ± SE.
Differences between experimental conditions were determined with an
unpaired Student’s t test. Body weights of mice before and
after dietary protocols were compared with a paired Student’s
t test. A value of P < 0.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To validate the quantitative RT-PCR approach, the results obtained by
Northern blot and quantitative RT-PCR analyses were compared. Figures 1
and 2
show the uroguanylin and guanylin mRNA expression normalized to GAPDH
mRNA expression for mouse intestine and kidney. Consistent with
previous findings (32), guanylin mRNA is expressed in the
intestine in a rostrocaudal gradient, ranging from very low in duodenum
to quite high in colon (Figs. 1 and 2B). In contrast, uroguanylin mRNA
is most prominent in proximal small intestine (Figs. 1 and 2A)
(32). In whole kidney extracts, a rather high uroguanylin
expression was found, levels being almost 50% of the maximal levels
found in intestine (in ileum), whereas the guanylin transcript was
barely detectable (Figs. 1 and 2).
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). In the intestine, the
cGMP-dependent effects of guanylin/uroguanylin are mediated by the
specific activation of GC-C and cGMP-dependent protein kinase type II
(cGKII). Starting from the same cDNAs used for the quantitative
analysis of uroguanylin/guanylin expression, we applied conventional
PCR to analyze the renal distribution of these signaling molecules.
GAPDH and GC-A were amplified simultaneously to confirm the efficiency
of the reaction. As shown in Fig. 3B, a prominent GC-C signal was
amplified from whole-kidney and colon extracts whereas the signal was
barely detectable in glomeruli and proximal tubules and undetectable in
the thick ascending limb of Henle’s loop and collecting ducts. A clear
signal for cGKII was obtained in all nephron segments. GC-A expression
was mainly found in glomeruli and collecting ducts.
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shows the water and food intakes as
well as body weights of mice subjected to different experimental
protocols. When wild-type control mice (GC-A +/+ and GC-C +/+,
respectively) were fed the low- or high-salt chow, their food intakes
and body weights remained stable. In the group of animals with
increased salt content in the food, water intake increased more than
2-fold. All of the mice (regardless of genotype) drinking the high-salt
(1% saline) water reduced their food-intake and lost a significant
amount of weight (Table 1), perhaps to stabilize the ratio of body
water to lean body mass (33). In water-deprived mice, food
intake slightly decreased and body weights were markedly reduced.
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shows the uroguanylin and
guanylin mRNA levels in these experimental groups, quantitated by
RT-PCR (LightCycler). Initial measurements showed that none of these
experimental conditions changed renal GAPDH gene expression, confirming
the suitability of GAPDH as a reference gene. Salt-restriction or
salt-loading via food did not alter renal uroguanylin and guanylin mRNA
expression. In mice receiving salt-loaded (1% saline) drinking water,
renal uroguanylin mRNA expression was increased about 1.8-fold;
guanylin expression levels were not affected (Fig. 4). These results
were further confirmed by northern-blot analysis (Fig. 5). Dehydration had no effect on renal
uroguanylin and guanylin expression (not shown).
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Under normal salt conditions, GC-A -/- mice exhibited a significantly higher systolic (148 ± 4.6 mmHg) and diastolic blood pressure (87 ± 5 mmHg, n = 9) than wild-type controls (109 ± 4 and 67 ± 3.2 mmHg). Arterial blood pressures in GC-C -/- mice were not different from wild-types (112 ± 3 and 69 ± 4 mmHg, n = 9). The high-salt treatment did not affect blood pressure in any genotype. The salt-resistance of the elevated blood pressure in GC-A -/- mice is in accordance with published data (20).
There were no significant differences between wild-types and GC-A -/-
mice with respect to renal uroguanylin expression after normal salt
diet (Fig. 6). In contrast, GC-C -/-
mice had significantly lower renal uroguanylin mRNA levels. All
genotypes showed significant, almost 2-fold elevations in renal
uroguanylin mRNA expression in response to high-salt (Fig. 6).
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). In contrast, CNP, guanylin,
uroguanylin, and nitric donors like sodium nitroprusside had no effect.
These results indicate that M-1 cells express membrane GC-A but not
GC-B, GC-C nor soluble GC.
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, there was a significant, up-to 3-fold
increase in the GAPDH-corrected uroguanylin PCR product in hypertonic
and dexamethasone-treated compared with control cells. Guanylin mRNA
remained undetectable in all tested conditions.
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Immunocytochemical detection of uroguanylin in M-1 cells
To examine the effects of high salt and/or dexamethasone on the
expression and localization of the uroguanylin propeptide in M-1 cells
we performed immunofluorescent staining followed by confocal microscopy
analysis. While unstimulated cells expressed only basal levels of
uroguanylin (Fig. 9A), a marked increase
in the intensity of the fluorescent signal could be observed in M-1
cells after incubation in high salt medium (Fig. 9C) or after
stimulation with dexamethasone (Fig. 9E) for 24 h. The
immunoreactivity was preferentially found in granules distributed
throughout the cytoplasm. To examine any effects of the treatment on
cell morphology and the cytoskeleton we stained actin filaments in the
same cultures with rhodamine-phalloidin (Fig. 9B, D, F, H). Prominent
actin bundles ran along the cell borders of the M-1 cells and smaller
stress fibers were spanning throughout the cytoplasm but no obvious
changes were apparent between the different treatments. Preabsorption
of the antiuroguanylin antibody with the peptide used for immunization
almost completely abolished the signal and showed only background
fluorescence in the FITC channel (Fig. 9G).
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) but not by Northern blot analysis (Fig. 10A).
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; only the full-length GC-C isoforms
are shown). To address this issue further, we carried out another
immunoblot with an antibody that recognizes a hydrophilic peptide on
the extracellular surface of GC-C. This antibody also recognized
full-length forms in the intestine as well as several smaller species
(data not shown). In contrast to the C-terminal specific antibody, this
extracellular antibody recognized a putative full-length GC-C isoform
that migrated to a similar position as intestinal GC-C. This molecular
species was enriched in kidney cortex (Fig. 10B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Guanylin and uroguanylin were initially isolated from rat jejunum and opossum urine, respectively (34, 35). Northern and RT-PCR analysis showed that the respective precursors are prominently expressed throughout the mucosa of the small and large intestine and, in very low levels, in extraintestinal tissues such as stomach, pancreas, heart, adrenals, and lung (32, 36, 37). In the digestive tract, uroguanylin is mainly stored in a special subset of endocrine, enterochromaffine cells of the small intestine, and guanylin in goblet and epithelial cells of the colonic mucosa (38, 39, 40). Both peptides are secreted into the intestinal lumen, to reach a specific receptor, guanylyl cyclase C (GC-C), in the apical, brush-border membrane of the intestinal epithelium. Binding to GC-C leads to increases in epithelial cGMP content and, via stimulation of a cGMP-dependent protein kinase (cGKII), to phosphorylation and activation of CFTR channels, with subsequent increases in Cl- and HCO3- secretion as well as decreased Na+ absorption (reviewed in Ref. 41).
Lower amounts of the guanylin and uroguanylin precursor forms are released from the intestine into the bloodstream and might exert extraintestinal effects (8, 9). In particular, synthetic guanylin and uroguanylin stimulate diuresis, natriuresis, and kaliuresis in vivo (in anesthetized mice and rats) and in vitro (in isolated perfused rat kidneys), uroguanylin being 10- to 100-fold more potent compared with guanylin (11, 12, 13). These observations led to the concept that guanylin and, in particular, uroguanylin, form a local, luminocrine system in the intestine, and an endocrine axis linking the intestine with the kidney. Thus, these peptides may regulate intestinal as well as renal electrolyte and water transport during postprandial periods of salt absorption by the digestive tract.
Along this line, it was demonstrated that low salt consumption significantly depresses the levels of uroguanylin and guanylin mRNA expression in the rat small intestine and colon (17, 42). The authors concluded that both pathways, suppression of guanylin-induced colonic Na+ efflux and of uroguanylin-induced natriuresis, constitute adaptative responses of the intestine to spare Na+ during periods of salt restriction.
As described above, these peptides are likely to regulate local water and electrolyte transport within the kidney itself. As shown in our study, renal uroguanylin mRNA expression is very prominent in mice, the ratios uroguanylin/GAPDH being about 50% of the ratios obtained in the small intestine. In contrast, guanylin mRNA contents in whole mouse kidney RNA extracts are barely detectable. Using isolated mouse nephron segments, we demonstrate by quantitative RT-PCR that these peptides display a differential distribution pattern along the nephron with uroguanylin being mainly expressed in the proximal tubules and guanylin mainly in the collecting ducts. This distribution and the fact that the kidney contains fewer collecting ducts than proximal tubules (in the mouse kidney cortex 8–10 PTs drain into one CD) may explain why in whole kidney extracts guanylin mRNA levels are much lower than uroguanylin levels. To examine prouroguanylin expression at the cellular level, we immunostained mouse kidney sections with a polyclonal antiuroguanylin antibody (30) (data not shown). Sensitivity and background problems made it difficult to precisely localize uroguanylin, although the intensity of staining in the cortical tubules appeared somewhat higher. These observations are in contrast with a recent study in the rat kidney, by Carrithers et al. (43). Using a semiquantitative RT-PCR approach these authors located guanylin/uroguanylin expression in all nephron segments, mainly in the loop of Henle and collecting tubules, suggesting that some species differences may exist for this peptide system.
Given the natriuretic and kaliuretic effects of these peptides, we investigated whether the renal expression is affected by changes in dietary salt or water intake. As described, we found that a high-salt load via drinking water significantly increases renal uroguanylin mRNA expression levels. In agreement with our observations, it has been reported that the 24-h urinary excretion of immunoreactive uroguanylin is increased in persons on a high-salt compared with a low-salt diet (44). Taken together, these responses suggest that stimulation of an intrarenal natriuretic system enables the kidney to concentrate and eliminate Na+ after an excessively increased oral salt load.
Interestingly, renal uroguanylin expression was modulated by high-salt loading via drinking water and not by increasing the salt content of the diet. As documented in the present and many published studies, increases in dietary salt lead to proportional increases in water intake, as a regulatory response allowing the increased production of urine and the urinary excretion of excess Na+ (33). In contrast, when salt is loaded via drinking water, there is a voluntary reduction of food intake in an amount such that body weight falls and the ratio of body water to lean body mass is conserved (33). A similar reaction was observed in mice subjected to dehydration: when water intake was restricted, food intake declined, again resulting in the loss of body weight (33). Because renal uroguanylin mRNA expression was increased by saline water but not dehydration, changes in uroguanylin mRNA expression cannot be explained simply in terms of reduced nutrient/body weight, but to excessive salt load.
Why is the renal uroguanylin expression modulated by salt-load via drinking water and not by dietary salt? The animals salt-loaded via drinking water are unable to compensate for elevated salt intake by drinking more water, as did the high dietary salt group. Thus, salt-intake through the drinking water causes a higher degree of salt loading. A greater impact of 1% NaCl in drinking water compared with 8% NaCl in diet on adaptative renal changes has been also observed in other studies (45).
A rather surprising observation in our study is the regulation of renal uroguanylin and not guanylin mRNA expression in response to a high-salt diet. At present time, we have no conclusive explanation for this result, although in context with the different cellular locations that we observed for both peptides in the kidney this seems to suggest different renal or downstream functions. Moreover, it is interesting to mention that patch-clamp experiments with isolated mouse collecting ducts demonstrated opposite effects on membrane voltages: guanylin caused depolarization and uroguanylin hyperpolarized these cells (46).
The abundant renal expression of uroguanylin prompted us to investigate the expression of GC-C and cGKII, the signaling molecules that mainly mediate uroguanylin and guanylin actions in the intestine. Whereas cGKII mRNA was easily detected by RT-PCR in all nephron segments, from proximal tubules to collecting ducts, only a very weak GC-C signal was detected by nested RT-PCR in glomeruli and proximal tubules. We extended these studies by examining the expression of GC-C in the rat using two well-characterized antibodies, one against the C-terminus of GC-C and the other against a hydrophilic peptide segment on the extracellular domain (30, 31). Although we were unable to detect GC-C with the C-terminal antibody, we were able to identify a band with the extracellular antibody that migrated to the same MW-position as intestinal GC-C. These discrepant results with a monospecific and highly sensitive C-terminal antibody and an extracellular antibody suggest either that (1) the processing of GC-C mRNA or protein differs at intestinal and renal sites or (2) the kidney expresses a distinct but highly homologous and comparably sized renal protein. The discrepancy between the renal amount of uroguanylin and GC-C at the mRNA level is also thought-provoking when combined with recent reports that some of the renal and intestinal effects of uroguanylin and guanylin persist in the GC-C deficient mouse (13). Could there be other as yet unidentified guanylyl cyclase or noncyclase receptors for these peptides? Moreover, does kidney uroguanylin signal not only intrarenally, but also at distal sites? Genetic data suggests that all of the mammalian guanylyl cyclases have been cloned (Garbers, D. L., Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX; personal communication); however additional analysis of the renal receptor for uroguanylin and guanylin as well as the fate of renal uroguanylin will be required to define the precise role of this intrarenal axis. In this context, one interesting observation in our study is that renal uroguanylin expression levels were significantly lower in GC-C-deficient mice, suggesting a mutual interaction between both, the peptide and this receptor, and a cGMP-dependent regulation of uroguanylin mRNA expression (positive feedback?).
In view of the complexity of the system in the intact animal, understanding of the mechanisms responsible for the regulation of renal mRNA expression may be facilitated by studies in cell culture model systems in which the effect of defined alterations can be assessed directly and in isolation. Because no cell culture model expressing uroguanylin or guanylin has been reported, we used PCR to screen different murine cell lines of renal origin. Our initial results showed that M-1 cells, originating from cortical collecting ducts and retaining many characteristics of the original cell types (22, 47), constitutively express uroguanylin mRNA. We did not detect guanylin mRNA in any of the tested cell lines.
Consistent with our in vivo observations, M-1 cells responded to hypertonic NaCl solutions with increased expression of uroguanylin mRNA. The same effect was also seen when mannitol was used as the osmotic agent. Also, dexamethasone increased uroguanylin expression in M-1 cells, what might be explained by a stimulatory effect of the glucocorticoid on Na+ uptake (47), or by direct regulation of gene transcription. These observations at the RNA level were strengthen by immunocytochemical studies, clearly showing increased storage of uroguanylin prohormone after exposure of M-1 cells to hypertonic NaCl or dexamethasone. Unfortunately, the expected changes in extracellular uroguanylin secretion could not be ascertained because of the lack of a suitable uroguanylin immunoassay. These data suggest that renal uroguanylin expression is directly influenced by renal hypertonicity and by its natriuretic/kaliuretic properties might contribute to stimulate renal saluresis in response to increased salt-load.
In view of the renal effects of uroguanylin and cardiac ANP, one could consider the possibility that both peptides participate in a synergistic or complementary fashion in the adaptation of kidney function to regulate salt and water homeostasis. This hypothesis prompted us to investigate the renal uroguanylin mRNA levels in mice with a genetic deletion of the ANP receptor (GC-A -/-) (20) and also whether ANP has a direct effect on uroguanylin expression in M-1 cells. As demonstrated, renal uroguanylin expression levels in GC-A -/- mice were not significantly different to wild-type mice, after both, normal-salt and a high-salt diet. M-1 cells do express GC-A, as demonstrated by RT-PCR (data not shown) and also by the stimulatory effects of synthetic ANP and BNP on intracellular cGMP levels; but ANP did not affect uroguanylin expression levels in M-1 cells. Thus, a modulatory effect of ANP on renal uroguanylin expression, either directly or via modulation of blood pressure and renal perfusion, seems unlikely.
In summary, quantitative real time RT-PCR constitutes a reproducible and sensitive mRNA quantification method, suitable to locate and monitor adaptative changes in regulatory genes with low expression level. Its application to cultured murine collecting duct (M-1) cells provided us with a useful model to extend our studies upon the regulation of uroguanylin mRNA expression in the kidney. Changes in uroguanylin synthesis and secretion by the renal tubular epithelium cells may contribute to the local control of cell function and ultimately to the adaptative responses of the kidney to dietary salt. The results of the present study corroborate the concept that the peptides guanylin and uroguanylin play an important role as endocrine as well as local systems involved in the adaptative responses of the intestine and kidney to changes in dietary salt load. It is conceivable that low-salt intake inhibits the intestinal systems guanylin (local?) and uroguanylin (endocrine?) and thereby leads to intestinal and renal Na+ conservation, whereas high-salt intake stimulates renal uroguanylin expression, which then contributes to increased renal natriuresis.
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Received November 6, 2000.
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