INTRODUCTION

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TWO SMALL (15 amino acid) peptides, named guanylin and uroguanylin, have been isolated and identified from rat intestine and opossum urine, respectively (4, 18). These small peptides are similar in structure to the peptide toxins secreted by enteric bacteria that causes traveler's diarrhea (Fig. 1). Guanylin, uroguanylin, and Escherichia coli heat-stable enterotoxins (ST) bind to and activate the apical membrane guanylate cyclase receptors found in the intestine, kidney, and other epithelia (4, 8, 13, 14, 18, 23). One of these membrane receptors for guanylin and uroguanylin has been identified by molecular cloning and named guanylate cyclase C (GC-C) (32). GC-C serves as a receptor for E. coli ST peptides in the lumen of the intestinal tract. The guanylin and uroguanylin peptides competitively inhibit ¹²⁵I-labeled ST binding to receptors on the surface of human T84 intestinal cells (4, 12, 18). Moreover, these peptides stimulate transepithelial Cl secretion in the T84 human colon carcinoma cell line and in the intestinal mucosa (4, 12, 16, 18, 22). The cGMP-mediated control of intestinal Cl and bicarbonate secretion may be important physiological actions for both guanylin and uroguanylin (4, 8, 16, 18). Currently, the physiological roles for guanylin and uroguanylin are poorly understood.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Primary structures comparing uroguanylin, guanylin, and bacterial heat-stable enterotoxins (ST). Single-letter code for amino acids is used.

Guanylin and/or its mRNA is found throughout the intestinal tract (4, 6, 19, 31, 33, 34). Molecular cloning of guanylin cDNAs reveals that the 15-residue form of guanylin that was isolated from the intestine is the COOH-terminal peptide contained within a larger precursor polypeptide, preproguanylin, consisting of 115-116 amino acids in the rat and human precursors (31, 33, 34). The highest levels of guanylin mRNA occur in the colon and distal small intestine of rats and man, whereas guanylin mRNA levels are highest in the ileum and jejunum of the opossum, compared with those observed in the colon and duodenum (6, 33, 34). Guanylin mRNA has also been identified in adrenal gland, kidney, and uterus/oviduct of rats (31, 33). Guanylin and proguanylin have also been isolated and identified from the intestinal mucosa of opossums (17-19). In addition, proguanylin circulates in the plasma of patients with chronic renal disease (24). Both ST and guanylin bind to cell surface receptors and raise the cellular cGMP levels in cultured T84 (intestinal cryptlike) cells (4, 17-19). Recently, we demonstrated that the lys-1 analogue of guanylin induces intestinal secretion in vitro, using the jejunum of rabbits in Ussing chambers, and lys-1 guanylin also elicited a natriuresis in the isolated perfused rat kidney (10).

Uroguanylin cDNAs have recently been isolated from opossum, human, and rat intestinal cDNA libraries (6, 21, 28, 29). The bioactive peptide, uroguanylin, is also found at the COOH terminus of preprouroguanylin, a 106- to 112-amino acid polypeptide in these species. Prouroguanylin and uroguanylin have been isolated from the intestinal mucosa of opossums, and these peptides are also found in the circulation of both the opossum and human subjects (6, 17-19, 20). Higher concentrations of bioactive uroguanylin are found in opossum, human, and rat urine, compared with the levels of guanylin (7, 18, 22). This suggests that uroguanylin may play a physiological role as an endocrine factor linking the intestinal tract with the kidney. In T84 colonic cells, uroguanylin appears to be ~10-fold more potent than guanylin in the activation of receptor guanylate cyclases that causes intracellular accumulation of the second messenger, cGMP (18). Uroguanylin, guanylin, and ST peptides interact with a common set of receptors in the intestine (4, 18, 17-19, 22). It seems likely that uroguanylin and/or guanylin may influence renal function in a manner consistent with the responses of the isolated perfused rat kidney to E. coli ST (26). This uroguanylin-like peptide was shown to be natriuretic, kaliuretic, and diuretic in this experimental model. Therefore, we investigated the effects of uroguanylin compared with guanylin on renal function in the perfused kidney. We found that uroguanylin stimulated increases in both urinary volume and the excretion of Na⁺ and K⁺ and that this peptide appeared to be more potent than guanylin in this kidney model.

	MATERIALS AND METHODS

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Guanylin and uroguanylin synthesis. The synthetic rat guanylin (PNTCEICAYAACTGC, mol wt 1516) and opossum uroguanylin (QEDCELCINVACTGC, mol wt 1596) were synthesized by a solid-phase method and purified as previously described (4, 18). The synthesis followed the solid-phase method with an Applied Biosystems 430A peptide synthesizer. Purification incorporated successive reverse-phase C₁₈ chromatography steps. The structures and purity of the synthetic peptide were verified by amino acid analysis, mass spectrometry, and NH₂-terminal sequence analysis.

Perfused rat kidney assay. Adult male Wistar rats weighing 250-350 g were anesthetized with pentobarbital sodium (5 mg/100 g body wt ip). Before the experiment, the animals were fasted for 24 h with access to water ad libitum. The perfusate was a modified Krebs-Henseleit solution with the following composition (in mM): 147 Na⁺, 5 K⁺, 2.5 Ca²⁺, 2.0 Mg²⁺, 110 Cl, 25 HCO₃, 1 SO²₄, and 1 PO³₄. Six grams of bovine serum albumin were added to the solution after a previous dialysis for 48 h at 4-6°C against a volume 10 times larger. Total perfusate used per experiment was 100 ml; immediately before the start of the perfusion, 100 mg of glucose, 50 mg of urea, and 50 mg of inulin were added to the perfusate. The pH was then adjusted to 7.4, and the solution was placed in the perfusion system.

The perfused rat kidney model followed the method of Bowman (1) as modified by Fonteles and others (9-11) by the introduction of a Silastic membrane oxygenator into the perfusion line. The system was calibrated for flow and resistance before each experiment. Perfusion pressure was measured at the tip of the stainless steel cannula. Perfusion pressure was allowed to fluctuate with experimental conditions but was maintained at 120 mmHg during the internal control. Each experiment was divided into four periods of 30 min each; these periods were further subdivided into equal intervals of 10 min. During each 10-min period, samples of perfusate and urine were collected for determinations.

A period of 15-20 min was allowed for blood washout when the kidney was first placed in the system. Guanylin and uroguanylin studies were initiated after an internal control of 30 min, and observations were made during the next 90 min. Samples were analyzed for Na⁺, K⁺, inulin, and osmolality. Clearance measurements were performed according to Martinez-Maldonado and Opava-Stitzer (27). Na⁺ and K⁺ were determined by flame photometry, and inulin was analyzed according to Fonteles et al. (9-11). Tissue K⁺ content of the perfused and unperfused kidneys was determined as previously described (15). Osmolality of the samples was measured in an Advanced Instruments osmometer (Needham Heights, MA). All chemicals were reagent grade and were acquired from either Sigma (St. Louis, MO) or Merck (West Point, PA).

A Macintosh Classic/80 computer system and software programs (KaleidaGraph and Statworks) were used for graphic calculations and data analyses. The data were presented as means ± SE. At least three different animals were used for each data point. Data were analyzed by a two-tailed Student's t-test when applicable and by analysis of variance. For statistical purposes, P  0.05 was considered significant.

	RESULTS

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Effects of guanylin. During the control experiments, kidneys were perfused for 2 h after an equilibration period of 15-20 min for adjustment of this preparation to the new perfusion conditions. Urine flow, glomerular filtration rate (GFR), and perfusion pressure were stable throughout the perfusion period with no statistically significant differences. Fractional excretion of Na⁺ and K⁺ in the urine were also maintained constant during the perfusion. Rat guanylin was administered as a bolus into the perfusate reservoir 30 min after the initial control period at concentrations of 0.33, 0.66, and 1.32 µM. Clearances and related biochemical measurements were made every 10 min and averaged for the groups of data at the 30-min intervals shown in Table 1. At the lowest concentration of guanylin tested (0.33 µM), no statistically significant differences from control were found in the values for urine flow, GFR, perfusion pressure, and percent of tubular Na⁺ reabsorption (%T_Na⁺). However, this dose of guanylin produced a remarkable decrease in percent of tubular K⁺ reabsorption (%T_K⁺) (P < 0.05). When 0.66 µM of guanylin was administered, there was no change in urine flow, GFR, and perfusion pressure, but guanylin produced the first significant decrease in Na⁺ reabsorption at the 120-min period, eliciting a decrease in %T_Na⁺ from 81.7 ± 2.4 to 77.2 ± 2.9 (P < 0.05). Urinary K⁺ excretion was significantly affected by guanylin (0.66 µM) with decreases in %T_K⁺ from 52.9 ± 8.5 during the control period to 39.5 ± 2.9, 27.8 ± 1.3 and 36.8 ± 3.7 at these time periods (P < 0.01). At the dose of guanylin of 1.32 µM, we observed no effects on urine flow and perfusion pressure. However, GFR increased significantly during the last experimental time period. %T_Na⁺ decreased from the internal control of 73.9 ± 8.5% to 64.5 ± 3.5%, with full recovery to the control values at the last time period. Guanylin elicited a kaliuresis as reflected by a decrease in %T_K⁺ from 61.9 ± 5.7% during the control period to 37.6 ± 3.8 and 22.5 ± 2.3% at 60 and 90 min (P < 0.01).

Effects of uroguanylin. This peptide was also administered as a bolus into the perfusate at concentrations of 0.19, 0.62, and 1.86 µM. We observed that all the doses of uroguanylin, except the lowest dose, promoted significant increases in urine flow (P < 0.05) in a dose-dependent manner (Fig. 2). However, significant increases in GFR were observed only with 0.62 and 1.86 µM concentrations of uroguanylin (P < 0.05) (Fig. 3). The doses of 0.62 and 1.86 µM also elicited small but significant increases in perfusion pressure (Table 2) (P < 0.05). Uroguanylin (0.62 µM) increased perfusion pressure from 115.5 ± 3.1 to 134.2 ± 3.4 mmHg (P < 0.05). The uroguanylin concentration of 1.86 µM caused no further increase in perfusion pressure, showing that a maximal effect was already reached. Uroguanylin elicited a natriuresis at all the concentrations used in these experiments (Fig. 4) (P < 0.05). We found no differences in the natriuretic responses to uroguanylin at 0.62 and 1.86 µM of this peptide, since both concentrations reduced tubular Na⁺ reabsorption from 78% to ~59% (P < 0.05) (Fig. 4). With respect to tubular K⁺ reabsorption, 0.62 µM of uroguanylin produced a smaller kaliuretic effect decreasing tubular K⁺ reabsorption, from 55.6 ± 1.4 to a maximal effect of 39.5 ± 2.6%, whereas, at 1.86 µM, this value decreased from 55.4 ± 4.6 to 26.4 ± 1.1% (P < 0.05) (Table 2). An important feature of these experiments with uroguanylin was the dose-related increase of osmolar clearance, with a maximal rise at the 1.86 µM dose of uroguanylin from control of 0.16 ± 0.01 to 0.34 ± 0.04 ml · g¹ · min¹ (Fig. 5).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Urine flow (UF) changes promoted by uroguanylin in the perfused rat kidney. Uroguanylin was added to the perfusate reservoir at concentrations shown. Results are expressed as means ± SE. * P < 0.05 compared with the 30-min internal control.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Glomerular filtration rate (GFR) changes promoted by uroguanylin in the perfused rat kidney; see Fig. 2 for other details. * P < 0.05 compared with the 30-min internal control.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Fractional sodium (%TNa+) changes promoted by uroguanylin in the perfused rat kidney; see Fig. 2 for other details. * P < 0.05 compared with the 30-min internal control.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Osmolar clearance (Cosm) changes promoted by uroguanylin in the isolated perfused rat kidney; see Fig. 2 for other details. * P < 0.05 compared with the 30-min internal control.

The overall results suggest that uroguanylin is more potent than guanylin in stimulating a natriuretic response (Fig. 6). Figure 7 suggests a higher kaliuretic effect promoted by guanylin. When similar concentrations of these peptides were used, uroguanylin (0.62 µM) elicited a greater increase in Na⁺ excretion than did guanylin (0.66 µM). The concentration of 0.33 µM guanylin had no effect on urinary Na⁺ excretion, whereas 0.19 µM uroguanylin caused a significant natriuresis. Due to the limited quantities of the synthetic peptides that were available, higher concentrations were not tested in this study. Thus it was difficult to estimate the relative potencies of guanylin compared with uroguanylin in the isolated perfused kidney.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Fractional sodium excretion (ENa+) promoted by different doses of guanylin and uroguanylin.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Fractional potassium excretion (EK+) promoted by different doses of guanylin and uroguanylin.

	DISCUSSION

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Our results demonstrate that uroguanylin and guanylin have natriuretic, kaliuretic, and diuretic activities in the isolated perfused kidney. These effects of uroguanylin and guanylin on renal function were predicted by earlier studies that found biochemical evidence for receptor guanylate cyclase signaling molecules in the kidney, as well as increases in the urinary excretion of cGMP, Na⁺, and K⁺ elicited by the administration of E. coli ST (13, 14, 23, 26). Discovery that the kidney was a target tissue for the ST peptides that cause secretory diarrhea in humans and other mammals predicted the existence of ST-like peptides in the body. Isolation of guanylin and uroguanylin from intestinal mucosa and urine fulfilled that prediction (4, 18). The new findings using the perfused rat kidney suggest that uroguanylin may be a major peptide that regulates the enzymatic activity of renal receptor guanylate cyclases physiologically, because uroguanylin appears to be more potent than guanylin in stimulating increases in urinary flow, as well as increasing Na⁺ and K⁺ excretion. Moreover, uroguanylin is abundant in urine compared with the low-to-nondetectable levels of guanylin in opossum, human, and rat urine (7, 18, 22). Prouroguanylin and uroguanylin have recently been isolated and identified from plasma, thus providing evidence that this peptide can serve in an endocrine pathway linking the intestine to the kidney (6, 20). Recent findings demonstrate uroguanylin and guanylin gene expression in renal tissues (28, 33). Thus an intrarenal signaling pathway for these peptides may be operating for the regulation of salt and water transport by cGMP under the influence of these endogenous peptides.

An intestinal natriuretic hormone was postulated to exist by Carey and colleagues (2, 25) as an explanation for the natriuresis that occurs following ingestion of a salt load. Administration of oral salt to humans or to experimental animals causes a much larger natriuresis than does the intravenous administration of saline. This implies that a natriuretic hormone could be released into the circulation from the intestine or other organs to stimulate the urinary excretion of NaCl. Uroguanylin may be involved in this mechanism that regulates kidney Na⁺ transport secondary to dietary salt. Of interest is the recent discovery that uroguanylin mRNA is expressed in atria and ventricles of the heart, as well as in the intestinal mucosa (6). These tissues may play a role in providing uroguanylin and/or prouroguanylin for secretion into the circulation. In this manner, the intestine and the myocardium could be linked with the kidney via uroguanylin as a novel natriuretic hormone. The long-lasting effects of uroguanylin and guanylin on urine Na⁺ excretion observed in this study contrasts with the shorter actions of atrial natriuretic peptides (ANPs) to influence renal Na⁺ excretion. Circulating ANPs do not appear to be influenced appreciably by oral salt loads; thus a role for these plasma salt-regulating hormones may not occur with respect to the natriuretic responses elicited by an oral salt load (30). However, the urinary excretion of a renal form of ANP is increased in human subjects in association with the natriuresis elicited by a high-salt meal (5). Perhaps uroguanylin and the renal form of ANP (urodilatin) peptides are involved in the physiological response of the kidney to an oral salt load, thus implicating an intrarenal paracrine mechanism involving ANP-like peptides and perhaps uroguanylin and/or guanylin produced in the kidney. In addition, circulating uroguanylin may participate in this regulatory mechanism because prouroguanylin and uroguanylin were isolated from plasma (6, 20).

Both uroguanylin and guanylin stimulated urinary K⁺ excretion, as demonstrated in the current experiments. Activation of renal receptors by ST and lys-1 guanylin also causes an increase in urinary K⁺ excretion (10, 26). However, the renal excretion of K⁺ can be influenced by different tubular mechanisms. The lowest dose of guanylin used in this study (0.33 µM) caused a significant kaliuresis without any effect on Na⁺ excretion. This suggests that guanylin may act on receptors in the distal nephron. The recent determination of the structure and the regulation of Kcn1 cGMP-gated K⁺ channels may provide an explanation for these effects of guanylin peptides on K⁺ transport in the perfused kidney (35). Although the nephron localization of receptors for these peptide agonists has not been described for the rat kidney, previous experiments with the opossum kidney have shown that receptors are present in the brush-border membranes of the proximal tubules (13, 14, 23). However, we cannot rule out that receptors for uroguanylin and guanylin may also occur in other segments of the nephron. It is reasonable to suggest that the natriuresis elicited by proximal actions of these peptides could produce a larger delivery of Na⁺ to the distal nephron, which may stimulate K⁺ secretion leading to the observed kaliuresis. The stimulation of K⁺ secretion by increased delivery of Na⁺ to the distal nephron may occur even when increases in urinary Na⁺ excretion are not observed. Future studies designed to elucidate the nephron location of target cells for uroguanylin and guanylin will be required to more completely assess the mechanism of action of these peptides in the kidney.

Uroguanylin elicited a significant increase in urine Na⁺ excretion at the lowest dose used (0.19 µM), but guanylin did not increase Na⁺ excretion at all time periods until a concentration of 1.3 µM was used. Higher doses of these peptides were not used in this study because of the limited availability of the synthetic peptides. Uroguanylin elicited a dose-dependent increase in urine Na⁺ excretion over the concentration range of 0.19-1.9 µM. Thus uroguanylin appears to be more potent than guanylin in the Na⁺ excretion response of the isolated perfused kidney. It has been shown that guanylin is degraded rapidly by chymotrypsin, whereas uroguanylin is resistant to this protease (3, 17, 19). Chymotrypsin-like proteases in the kidney tubules may degrade guanylin, which could contribute to its apparently lower potency, relative to uroguanylin as natriuretic peptide agonists in this model system. Degradation of guanylin by renal proteases could also explain the low-to-nondetectable levels of bioactive guanylin found in mammalian urine (7, 18, 22).

In summary, treatment of the isolated perfused kidney with uroguanylin and guanylin resulted in a clear increase in the urinary excretion of Na⁺, K⁺, and water. Therefore, we postulate that these peptides may influence salt and water homeostasis by some physiological interactions in the kidney. Uroguanylin and guanylin may participate in the regulation of salt and water transport in both the intestine and the kidney.