Guanylyl cyclase-C receptor mRNA distribution along the rat nephron

doi:10.1016/S0167-0115(00)00139-7

Regulatory Peptides

Volume 95, Issues 1–3, 24 November 2000, Pages 65-74

https://doi.org/10.1016/S0167-0115(00)00139-7 Get rights and content

Abstract

Guanylin (GN) and uroguanylin (UGN) are two recently identified peptides that have been shown to affect water and electrolyte transport in both the intestine and the kidney. Mechanistically, the effects of both peptides are thought to be mediated by intracellular cGMP which results from ligand binding to a plasma membrane guanylyl cyclase-C (GC-C) receptor. To date, the specific intrarenal site(s) of GN and UGN action have not been established. To begin to address this issue, the present studies utilized semi-quantitative RT-PCR to assess the distribution of GC-C mRNA in specific microdissected segments of the rat nephron. GC-C mRNA expression was highest in the cortical collecting tubule, followed by the proximal convoluted tubule, medullary thick ascending limb and collecting tubule, and thin limbs of Henle’s loop. Expression levels were significantly lower in all other segments tested, including the glomerulus. The renal tubular expression pattern for cGMP-dependent protein kinase II (cGK-II) mRNA, which is activated in response to GN/UGN-dependent cGMP accumulation, was similar to that for GC-C. Notably, both GN and UGN mRNAs were also expressed along the nephron. The highest levels of expression for both peptides were detected in the medullary collecting tubule. Lower, but comparable levels of GN and UGN expression also occurred in the cortical collecting tubule, cortical and medullary thick ascending limb, and thin limbs of Henles loop. In the proximal convoluted tubule, GN mRNA expression was also quite high, while UGN mRNA was almost undetectable. The presence of renal GC-C and cGK-II in the kidney are consistent with a proposed endocrine function for GN and UGN. In addition however, the present data suggest that intrarenally synthesized GN and UGN may also contribute to the regulation of renal tubular transport.

Introduction

Guanylin and uroguanylin are small, heat-stable peptides that were originally isolated from the small intestine and urine, respectively [1], [2], [3]. Both peptides are synthesized as inactive precursor prohormones by cells within the gastrointestinal mucosa and secreted into the lumen, where they are then cleaved to their 15–16 amino acid bioactive moieties [4], [5]. Functionally, it has been proposed that guanylin and uroguanylin can regulate intestinal salt and water transport during digestion. Additionally, these peptides can stimulate HCO₃⁻ secretion in order to neutralize lumenal HCl in the duodenum [6], [7], and organic acids derived from enteric bacteria in the large intestine [8], [9]. The intracellular mechanism of guanylin and uroguanylin action is mediated by cyclic-3′,5′-guanosine monophospate (cGMP) which specifically activates cGMP-dependent protein kinase II (cGK-II) [9], [10], [11]. To date, only one plasma membrane receptor for guanylin and uroguanylin has been functionally identified and cloned from mammalian species, termed guanylyl cyclase C (GC-C, gucy2c) [12], [13]. Pathophysiologically, several strains of bacteria including Escherichia coli elaborate heat-stable enterotoxins (STa) which also bind to intestinal GC-C receptors, and cause severe secretory diarrhea [11], [14].

In addition to their established effects on the GI tract, several recent studies have suggested that guanylin and uroguanylin can also affect renal function [11], [15], [16]. In both the isolated perfused rat kidney [17], [18], and in the sealed-urethra mouse model [15], [19] guanylin and uroguanylin administration significantly increased urinary volume as well as sodium and potassium excretion. Coincident with these solute excretory patterns, urinary cGMP excretion also increased after peptide administration.

These data have led to the hypothesis that the renal effects of guanylin/uroguanylin that are synthesized in the GI tract may represent a novel endocrine pathway involved in the maintenance of salt and water homeostasis. Although previous studies have reported ¹²⁵I-guanylin [20] and ¹²⁵I-ST binding [21], [22], [23], and GC-C mRNA expression [24], [25], [26] in heterogeneous whole kidney preparations, the specific intrarenal site(s) of guanylin and uroguanylin action have yet to be fully established. Therefore, the principal aim of the present study was to apply RT-PCR to individual microdissected nephron segments to identify the distribution pattern for GC-C mRNA.

While an intestinal site of synthesis and a renal site of action for these peptides is consistent with a classic endocrine signaling pathway, there is evidence that guanylin and uroguanylin may also be synthesized intrarenally [24], [26], [27], [28]. Therefore, the present study was also designed to identify specific sites of guanylin/uroguanylin mRNA expression along the nephron, which would support the existence of an additional paracrine axis for regulation of renal function by these peptides.

Section snippets

Renal tubule microdissection

Male Sprague–Dawley rats (200–250 g) were allowed free access to standard laboratory rat chow and tap water prior to study. Individual nephron segments were microdissected from collagenase-digested kidneys using techniques described in detail previously [29], [30]. Briefly, under sodium pentobarbital anesthesia (40 mg/kg i.p.), the left kidney was selectively perfused with 10 ml of HBSS containing 0.1% collagenase, 0.1% hyaluronidase and 0.1% bovine serum albumin (collagenase medium). Thin

Renal tubular distribution of guanylyl cyclase C mRNA

Southern analysis of the RT-PCR ethidium bromide-stained gels demonstrated specific binding of the GC-C probe to a 400-bp amplification product in all nephron segments tested (Fig. 2). To standardize the RT-PCR procedure and to confirm efficient reverse transcription of tissues, GC-C and β-actin were amplified simultaneously under identical conditions (Fig. 2). The amplification of GC-C in each nephron segment was then normalized by the β-actin signal (GC-C signal/β-actin signal). The strongest

Discussion

Several recent studies have suggested that the newly identified intestinal peptides guanylin and uroguanylin increase renal salt and water excretion in mammals [15], [17], [18], [19]. However, the potential intrarenal site(s) of action of these peptides have not been investigated. Based on semi-quantitative RT-PCR analysis of mRNA levels for the GC-C receptor, our data suggest that these peptides may affect transport in several nephron segments, particularly the proximal convoluted tubule,

Acknowledgments

The authors would like to thank the technical assistance of Jason J. Chang and the intellectual contribution and advise of Leonard R. Forte, Ph.D., University of Missouri, Truman VA Medical Center and Andre F. Carvalho, Federal University of Ceara, Brazil. This work was supported by the Medical Research Service of the Department of Veterans Affairs, Lexington, Kentucky (RNG, BAJ, SLC), the National American Heart Association (BAJ), and the Ohio Affiliate of the American Heart Association (BAJ,

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Effect of renoguanylin on hydrogen/bicarbonate ion transport in rat renal tubules
2009, Regulatory Peptides
Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 μM and 10 μM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 μM concentrations. Comparing control and REN concentration of 1 μM, JHCO₃⁻, nmol cm^− 2 s^− 1 − 1,76 ± 0,11_control × 1,29 ± 0,08_{REN 10 μM}; P < 0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 μM (JHCO₃⁻, nmol cm^− 2 s⁻1 − 0.80 ± 0.07_control × 0.60 ± 0.06_{REN 1 μM}; P < 0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na⁺/H⁺exchanger and H⁺-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway.
Uroguanylin and Guanylin: Endocrine Link Connecting the Intestine and Kidney for Regulation of Sodium Balance
2008, Seldin and Giebisch's The Kidney
Guanylin and uroguanylin are small peptides that are produced in the intestinal mucosa and regulate cellular function through activation of receptor-guanylate cyclase signaling molecules locally in target cells of intestine and at a distance in the body via endocrine pathways. Their discovery stems from studies of bacterial toxins implicated in mechanisms underlying a disorder given the trivial name, traveler's diarrhea. This cholera-like disease is associated with intense diarrhea with marked loss of fluid and electrolytes induced by bacterial peptides that pathologically mimic the actions of guanylin and uroguanylin. However, physiological roles of the natural peptide hormones involve regulation of body sodium balance in times when animals are exposed to excess levels of sodium chloride in the environment or in the diet. Discovery of an orphan receptor-guanylate cyclase for microbial toxins and demonstration that toxin peptides elicit strong natriuretic responses in the isolated perfused kidney opened up a new area of endocrinology involving the peptide hormones, uroguanylin and guanylin. These intestinal natriuretic hormones may act physiologically in the body by a novel endocrine axis linking the intestine and kidney for regulation of body sodium homeostasis. Administration of oral salt loads in vivo elicit marked increases in urinary salt excretion that are uroguanylin-dependent because transgenic uroguanylin^−/– mice exhibit impaired natriuretic responses to oral salt loads and have elevated blood pressure. High-salt diets in mammals or adaptation to seawater in the eel stimulate uroguanylin and guanylin mRNA expression in the intestine. The intestine responds to salt with increased secretion of both peptides. High salt also increases uroguanylin mRNA levels in the kidney. Therefore, uroguanylin is likely to serve in an endocrine link between the intestine and kidney, whereas this peptide could also act locally within the kidney as a physiological means of regulating body sodium balance. Additional evidence for uroguanylin's influence on sodium balance is derived from experiments of nature where it has been shown that sodium retention secondary to either heart failure or the nephrotic syndrome is associated with marked increases in uroguanylin levels in vivo. These findings reveal that uroguanylin functions as a counterregulatory hormone in physiological states associated with excess body sodium levels. Finally, we propose that uroguanylin and guanylin may also act to regulate salt excretion by the intestine in times when animals are exposed to high levels of salt in their environment. The physiological actions that regulate body sodium balance may have been important selective pressures guiding the evolution of uroguanylin and guanylin, thus maintaining their distinctive structures and activities for about 400 million years that separate fish and man.
Uroguanylin And Guanylin. Endocrine Link Connecting The Intestine And Kidney For Regulation Of Sodium Balance.
2007, Seldin and Giebisch's The Kidney: Physiology & Pathophysiology 1-2
Guanylin and uroguanylin are small peptides that are produced in the intestinal mucosa and regulate cellular function through activation of receptor-guanylate cyclase signaling molecules locally in target cells of intestine and at a distance in the body via endocrine pathways. Their discovery stems from studies of bacterial toxins implicated in mechanisms underlying a disorder given the trivial name, traveler's diarrhea. This cholera-like disease is associated with intense diarrhea with marked loss of fluid and electrolytes induced by bacterial peptides that pathologically mimic the actions of guanylin and uroguanylin. However, physiological roles of the natural peptide hormones involve regulation of body sodium balance in times when animals are exposed to excess levels of sodium chloride in the environment or in the diet. Discovery of an orphan receptor-guanylate cyclase for microbial toxins and demonstration that toxin peptides elicit strong natriuretic responses in the isolated perfused kidney opened up a new area of endocrinology involving the peptide hormones, uroguanylin and guanylin. These intestinal natriuretic hormones may act physiologically in the body by a novel endocrine axis linking the intestine and kidney for regulation of body sodium homeostasis. Administration of oral salt loads in vivo elicit marked increases in urinary salt excretion that are uroguanylin-dependent because transgenic uroguanylin^−/– mice exhibit impaired natriuretic responses to oral salt loads and have elevated blood pressure. High-salt diets in mammals or adaptation to seawater in the eel stimulate uroguanylin and guanylin mRNA expression in the intestine. The intestine responds to salt with increased secretion of both peptides. High salt also increases uroguanylin mRNA levels in the kidney. Therefore, uroguanylin is likely to serve in an endocrine link between the intestine and kidney, whereas this peptide could also act locally within the kidney as a physiological means of regulating body sodium balance. Additional evidence for uroguanylin's influence on sodium balance is derived from experiments of nature where it has been shown that sodium retention secondary to either heart failure or the nephrotic syndrome is associated with marked increases in uroguanylin levels in vivo. These findings reveal that uroguanylin functions as a counterregulatory hormone in physiological states associated with excess body sodium levels. Finally, we propose that uroguanylin and guanylin may also act to regulate salt excretion by the intestine in times when animals are exposed to high levels of salt in their environment. The physiological actions that regulate body sodium balance may have been important selective pressures guiding the evolution of uroguanylin and guanylin, thus maintaining their distinctive structures and activities for about 400 million years that separate fish and man.
Interaction of atrial natriuretic peptide, urodilatin, guanylin and uroguanylin in the isolated perfused rat kidney
2006, Regulatory Peptides
Escherichia coli heat-stable enterotoxin (STa), guanylin and uroguanylin are novel natriuretic and kaliuretic peptides that bind to and activate membrane guanylate cyclase (GC) receptors such as GC-C and OK-GC that are expressed in the kidney and intestine. Atrial natriuretic peptide (ANP) and its renal form (urodilatin, UROD) elicit natriuretic effects by activation of a different membrane guanylate cyclase, GC-A. Experiments were done in perfused rat kidneys to search for possible synergistic interactions between ANP, UROD, guanylin and uroguanylin on renal function. Pretreatment with ANP (0.03 nM) enhanced guanylin (0.19 μM) natriuretic activity (%ENa⁺; from 18.5 ± 4.25 to 31.5 ± 1.69, P < 0.05, 120 min) and its kaliuretic activity (%EK⁺; from 24.5 ± 4.43 to 50.6 ± 3.84, P < 0.05, 120 min). Furthermore, ANP increased the natriuretic (29.05 ± 3.00 to 37.8 ± 2.95, P < 0.05, 120 min) and kaliuretic (from 33.2 ± 3.52 to 42.83 ± 2.45, P < 0.05, 120 min) responses of perfused kidneys treated with low-dose (0.06 μM) uroguanylin. In contrast, ANP clearly inhibited the uroguanylin-induced (0.31 μM) increase in %ENa⁺ (from 35.9 ± 2.37 to 14.8 ± 1.93, P < 0.05, 120 min), and in %EK⁺ (from 51.0 ± 4.43 to 38.8 ± 3.61, P < 0.05, 120 min). UROD (0.03 nM) also enhanced the guanylin-induced natriuresis (to %ENa⁺ = 31.0 ± 1.93, P < 0.05, 120 min) and kaliuresis (to %EK⁺ = 54.2 ± 3.61, P < 0.05, 120 min), and inhibited the %ENa⁺ of uroguanylin (0.31 μM) to 17.9 ± 1.67 as well as its %EK⁺ to 24.3 ± 3.13 (both at 120 min, P < 0.05). The synergism between ANP and UROD with either guanylin or uroguanylin at sub-threshold doses and the unexpected antagonism between ANP and UROD with uroguanylin at a pharmacological dose point to possible interactions between natriuretic peptide receptor (NPR) and uroguanylin/guanylin receptor signaling pathways. The interactions herein described may play a contributory role in the regulation of kidney function in many pathophysiological states, such as in the saliuresis following ingestion of salty meals.
Uroguanylin and guanylin peptides: Pharmacology and experimental therapeutics
2004, Pharmacology and Therapeutics
Guanylin, uroguanylin, and the bacterial heat-stable enterotoxin (ST) peptides comprise a new family of cyclic guanosine 3′-5′ monophosphate (cGMP)-regulating agonists. The discovery of guanylin and uroguanylin peptides stems from studies of cellular mechanisms underlying a form of secretory diarrhea caused by enteric bacteria. Guanylin, uroguanylin, and microbial ST peptides activate a common apical membrane receptor-guanylate cyclase (R-GC) that elicits large increases in the intestinal secretion of chloride and bicarbonate via the intracellular second messenger, cGMP. Guanylin and uroguanylin were isolated from rat jejunum and opossum urine, respectively. These peptides are endogenous peptide hormones that physiologically regulate R-GC signaling proteins in target cells. Physiological roles for these peptides include the regulation of epithelial cell balance in the intestinal epithelium and modulation of sodium balance through actions in the kidney. The guanylin-uroguanylin-ST peptides are candidate therapeutic agents targeting receptors in the intestine, kidney, and other epithelia. For example, uroguanylin has anti-tumor actions in an animal model for human colon cancer. The ST peptides can be used as diagnostic agents to detect secondary colon cancers by single photon-emitting computed tomography (SPECT) imaging, thus localizing metastatic forms of colon cancer. Other examples of potential therapeutic applications for the guanylin family of cGMP-regulating agonists are: (1) the irritable bowel syndrome (IBS) with constipation, (2) salt-dependent forms of high blood pressure, (3) liver regeneration and repair, and (4) respiratory diseases such as asthma. Competitive pharmacological antagonists of bacterial ST peptides offer a means for treating the diarrhea caused by ST-secreting strains of enteric bacteria.
Guanylin and uroguanylin induce natriuresis in mice lacking guanylyl cyclase-C receptor
2004, Kidney International
Citation Excerpt :
First, it has yet to be established where functional GC-C or alternate GN/UGN receptors are located within specific nephron segments. Our previous data would suggest that primary sites of action would include the proximal convoluted tubule, medullary thick ascending limb, and the cortical collecting tubules as targets for GN, UGN, and STa [32]. Thus, further detailed segmental analysis employing in vivo micropunture and in vitro microperfusion are necessary to identify specific nephron segments containing functional GN/UGN/STa sites of action.
Guanylin and uroguanylin induce natriuresis in mice lacking guanylyl cyclase-C receptor.
Guanylin (GN) and uroguanylin (UGN) are intestinally derived peptide hormones that are similar in structure and activity to the diarrhea-causing Escherichia coli heat-stable enterotoxins (STa). These secretagogues have been shown to affect fluid, Na⁺, K⁺, and Cl⁻ transport in both the intestine and kidney, presumably by intracellular cyclic guanosine monophosphate (cGMP)-dependent signal transduction. However, the in vivo consequences of GN, UGN, and STa on renal function and their mechanism of action have yet to be rigorously tested.
We hypothesized that intravenous administration of GN, UGN, or STa would cause an increase in natriuresis in wild-type mice via cGMP and guanylyl cyclase-C (GC-C, Gucy2c), the only known receptor for these peptide-hormones, and that the peptide-induced natriuresis would be blunted in genetically altered mice devoid of GC-C receptors (GC-C^(−/−) null).
In wild-type mice using a modified renal clearance model, GN, UGN, and STa elicited significant natriuresis, kaliuresis, and diuresis as well as increased urinary cGMP levels in a time- and dose-dependent fashion. Absolute and fractional urinary sodium excretion levels were greatest ∼40 minutes following a bolus infusion with pharmacologic doses of these peptides. Unexpectedly, GC-C^(−/−) null mice also responded to the GN peptides similarly to that observed in wild-type mice. Glomerular filtration rate (GFR), blood pressure, and plasma cGMP in the mice (wild-type or GC-C^(−/−) null) did not significantly vary between the vehicle- and peptide-treatment groups. The effects of UGN may also influence long-term renal function due to down-regulation of the Na⁺/K⁺ ATPase γ-subunit and the Cl⁻ channel ClC-K2 by 60% and 75%, respectively, as assessed by differential display polymerase chain reaction (PCR) (DD-PCR) and Northern blot analysis of kidney mRNA from mice treated with UGN.
GN, UGN, and STa act on the mouse kidney, in part, through a cGMP-dependent, GC-C–independent mechanism, causing significant natriuresis by renal tubular processes. UGN may have further long-term effects on the kidney by altering the expression of such transport-associated proteins as Na⁺/K⁺ ATPase and ClC-K2.

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Guanylyl cyclase-C receptor mRNA distribution along the rat nephron

Abstract

Introduction

Section snippets

Renal tubule microdissection

Renal tubular distribution of guanylyl cyclase C mRNA

Discussion

Acknowledgments

Gastroenterology

Adv Pharmacol

Am J Kidney Dis

Cell

Biochem Biophys Res Commun

Reg. Pept.

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Mol. Cell Endocrinol.

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