Increases in Guanylin and Uroguanylin in a Mouse Model of Osmotic Diarrhea Are Guanylate Cyclase C Independent

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1 GASTROENTEROLOGY 2001;121: Increases in Guanylin and Uroguanylin in a Mouse Model of Osmotic Diarrhea Are Guanylate Cyclase C Independent KRIS A. STEINBRECHER,* ELIZABETH A. MANN, RALPH A. GIANNELLA, and MITCHELL B. COHEN* *Division of Pediatric Gastroenterology, Hepatology and Nutrition and Graduate Program in Molecular and Developmental Biology, Children s Hospital Research Foundation, Cincinnati, Ohio; and Division of Digestive Diseases, Veterans Medical Center, University of Cincinnati, Cincinnati, Ohio Background & Aims: Guanylin and uroguanylin are peptide hormones that are homologous to the diarrheacausing Escherichia coli enterotoxins. These secretagogues are released from the intestinal epithelia into the intestinal lumen and systemic circulation and bind to the receptor guanylate cyclase C (GC-C). We hypothesized that a hypertonic diet would result in osmotic diarrhea and cause a compensatory down-regulation of guanylin/uroguanylin. Methods: Gut-to-carcass weights were used to measure fluid accumulation in the intestine. Northern and/or Western analysis was used to determine the levels of guanylin, uroguanylin, and GC-C in mice with osmotic diarrhea. Results: Wild-type mice fed a polyethylene glycol or lactose-based diet developed weight loss, diarrhea, and an increased gut-tocarcass ratio. Unexpectedly, 2 days on either diet resulted in increased guanylin/uroguanylin RNA and prohormone throughout the intestine, elevated uroguanylin RNA, and prohormone levels in the kidney and increased levels of circulating prouroguanylin. GC-C deficient mice given the lactose diet reacted with higher gut-to-carcass ratios. Although they did not develop diarrhea, GC-C sufficient and deficient mice on the lactose diet responded with elevated levels of guanylin and uroguanylin RNA and protein. A polyethylene glycol drinking water solution resulted in diarrhea, higher gutto-carcass ratios, and induction of guanylin and uroguanylin in both GC-C heterozygous and null animals. Conclusions: We conclude that this model of osmotic diarrhea results in a GC-C independent increase in intestinal fluid accumulation, in levels of these peptide ligands in the epithelia of the intestine, and in prouroguanylin in the kidney and blood. Secretion or absorption of water through the epithelial layer of the gastrointestinal tract occurs in response to shifts in osmotic gradients that are controlled through activation of membrane-bound ion channels and transporters. Hypertonic luminal contents are brought to near isotonicity in the proximal small intestine via paracellular water movement through tight junctions. Osmotic diarrhea occurs when the absorptive capacity of the small and large intestine is exceeded by the fluid that enters the lumen in response to a hypertonic solution. The intestine actively responds to osmotic diarrhea by increasing its absorptive capacity in an attempt to eliminate water loss and eventual dehydration. In contrast to these absorptive mechanisms, much less is known about the regulation of intestinal secretory pathways in response to diarrheal states. One recently characterized intestinal secretory pathway is mediated through the guanosine 3,5 -cyclic monophosphate (cgmp)-dependent activation of the cystic fibrosis transmembrane conductance regulator (CFTR). Increases in cgmp lead to phosphorylation of the CFTR by the membrane-bound cgmp-dependent protein kinase II (cgk II) and release of chloride and/or bicarbonate from the cell as well as CFTR-mediated modulation of transmembrane ion channel conductance. 1 5 CFTR activation is critical for proper extracellular hydration and electrolyte balance and its loss results in significant gastrointestinal and pulmonary disease. Pathologic levels of fluid secretion through this pathway are caused by the heat stable enterotoxin, STa. 6 Upon elaboration by Escherichia coli, STa binds to a transmembrane guanylate cyclase receptor, guanylate cyclase C (GC-C), and acts as a superagonist. 7 Activation of GC-C by STa results in high levels of intracellular cgmp and culminates in increased fluid secretion into the intestine. 1,8 The secretory diarrhea mediated by this toxin is the cause of significant infant mortality and morbidity in developing countries. Abbreviations used in this paper: CFTR, cystic fibrosis transmembrane conductance regulator; DRA, down-regulated in adenoma; G/C, gut-to-carcass (ratio); GC-C, guanylate cyclase C; NHE3, sodium-hydrogen exchanger by the American Gastroenterological Association /01/$35.00 doi: /gast

2 1192 STEINBRECHER ET AL. GASTROENTEROLOGY Vol. 121, No. 5 Two endogenous peptide ligands, guanylin and uroguanylin, also bind to and activate GC-C. 9,10 In most mammalian species, these proteins are produced predominately in the intestine, although uroguanylin is also expressed in the kidney Proguanylin and prouroguanylin are secreted into the intestine and blood stream as 11-kilodalton prohormones and are each cleaved to release the active 15 amino acid carboxy termini that bind to GC-C Because guanylin and uroguanylin are highly homologous to STa, activate the same pathway, and cause increased short-circuit current in intestinal epithelial cells, it has been suggested that they play a role in fluid secretion in the intestine. Circulating uroguanylin causes natriuresis, kaliuresis, and diuresis in isolated and perfused rat kidneys, and it has been suggested that uroguanylin represents a gut-to-kidney signaling hormone that, upon ingestion of high-salt meals, causes natriuresis in anticipation of increased intestinal salt absorption. 17,18 Other studies of the function of guanylin and uroguanylin suggest that these peptides may have other physiological roles as well We sought to develop an animal model to investigate regulation of guanylin/uroguanylin production. Because guanylin and uroguanylin are putative intestinal secretagogues, we postulated that sustained fluid loss into the intestine would down-regulate guanylin and uroguanylin levels. Here, we establish a model of osmotic diarrhea in mice using nonabsorbable osmolytes and demonstrate that this model causes weight loss, liquid stool, and increases in compensatory absorptive mechanisms in the intestine. Contrary to our expectations, we show that guanylin and uroguanylin are substantially increased at the RNA and prohormone levels and that their receptor, GC-C, is unchanged. We show that both serum and kidney levels of prouroguanylin are elevated. Furthermore, we show that these increases are not dependent on the presence of GC-C because GC-C null animals also respond with increased levels of guanylin and uroguanylin. Materials and Methods Lactose Diet Administration All animal studies were approved by the Institutional Animal Care and Use Committee of the Cincinnati Children s Hospital Medical Center. Eight- to 10-week-old wild-type FVB/N female mice (Jackson Laboratory, Bar Harbor, ME) were housed in microisolator cages, exposed to standard 12- hour light and 12-hour dark cycles. GC-C null homozygous and heterozygous littermate animals of C57Bl/6 background were maintained under similar conditions. 24 After initial body weight determination, mice were given a powdered control diet or a mixture consisting of 75% D-lactose/25% control chow for 2 or 4 days. The control diet was made according to Soleimani et al. 25 Mice were allowed water ad libitum during the study period. Animals were killed during a 2-hour period on days 2 or 4 to avoid differences in circadian expression of guanylin and uroguanylin between control and lactose-fed groups. 26 Mice were anesthetized, weighed, and blood was collected from the inferior vena cava. The intestinal tract was then removed and weighed as a whole (duodenum to distal colon). The weight of the animal carcass was also recorded and the ratio of gut-to-carcass (G/C) weight was calculated. For protein and messenger RNA (mrna) studies, the intestine was flushed with cold saline, separated into segments, and stored at 80 C. Intestinal segments were defined as follows: proximal jejunum (proximal third of the small intestine), ileum (distal third of the small intestine), cecum, proximal colon (proximal 40% of colon), and distal colon (distal 60% of the colon). Kidneys were also removed, rinsed, and stored at 80 C for later study. Some studies were performed using, as the sole water source, a solution of 40 mmol/l polyethylene glycol (PEG) 3350 (Sigma, St. Louis, MO). Animals were provided the PEG 3350 solution for 48 hours and tissue samples were processed as above. Northern Blot Analysis Frozen tissue was pulverized in chilled mortars and pestles and RNA was extracted using Trizol Reagent (Gibco BRL, Gaithersburg, MD) according to the manufacturer s suggested protocol. Total RNA (20 g per sample) was fractionated using formaldehyde-agarose gel electrophoresis and blotted onto Magnacharge nylon membrane (Osmonics, Westborough, MA) using standard capillary transfer methods. Portions of the guanylin, uroguanylin, GC-C, down-regulated in adenoma (DRA) and sodium-hydrogen exchanger 3 (NHE3) complementary DNA (cdnas) were radiolabeled with [ - 32 P] (DuPont-NEN, Boston, MA) using the Random Primer Labeling system (Roche Molecular Biochemicals, Indianapolis, IN) as described previously. 11,27 Some blots were stripped with 0.1% sodium dodecyl sulfate, 0.1 standard saline citrate before being probed again. Rat NHE3 and mouse DRA cdna probe templates were generously provided by Dr. Gary Shull, University of Cincinnati. 28,29 All blots were then stripped and hybridized with a 32 P-radiolabeled 18S ribosomal subunit probe to normalize cdna probe signals. Normalized RNA data were determined by dividing the cdna signal by the 18S signal in each lane. Within each time point, the greatest normalized tissue signal mean was then arbitrarily set at 100 and all other tissues were adjusted accordingly. Blots were visualized and quantitated using a Molecular Dynamics PhosphorImager system (Molecular Dynamics, Sunnyvale, CA). Western Blot Analysis Tissue was homogenized (approximately 100 mg tissue in 1 ml buffer) in 2 mmol/l Tris-HCL and 50 mmol/l mannitol buffer containing protease inhibitor (Sigma-Aldrich, St. Louis, MO) and centrifuged at 16,000g for 20 minutes. Supernatants were collected and quantitated using the Brad-

3 November 2001 GUANYLIN AND UROGUANYLIN IN OSMOTIC DIARRHEA 1193 ford method. Blood samples were centrifuged at 12,000g for 10 minutes and sera were obtained. Tissue (65 g) and serum (2.5 L) samples were electrophoresed using NuPAGE 4% 12% gradient polyacrylamide gels (Invitrogen, Carlsbad, CA) and electroblotted onto nitrocellulose membranes according to the manufacturer s protocol. Membranes were immunoblotted as previously described using a 1:1000 dilution of antisera that recognize proguanylin (antibody 2538) and prouroguanylin (antibody 6910). These antibodies were generously provided by Dr. Michael Goy of the University of North Carolina and have been validated and described previously. 30,31 After incubation with a horseradish peroxidase conjugated secondary antibody, signal was visualized on Kodak X-OMAT AR film (Kodak, Rochester, NY) using a commercially available chemiluminescent kit (NEN Life Science Products, Boston, MA). Statistical Analysis All values are presented as means SE. All comparisons are made between animals on control diet and animals on osmotic diet within the same time point using the unpaired t test. Differences were considered statistically significant at P 0.05 unless otherwise stated. Results Effects of the Lactose-Based Diet on Wildtype FVB/N and GC-C Deficient Mice To cause osmotic diarrhea, female FVB/N mice were fed a lactose-containing diet for 2 or 4 days. The animals consumed both control and lactose diets readily and remained active and alert during the study period. At day 2, the lactose-fed group had severe diarrhea that resulted in wet, discolored anogenital areas and evident liquid stool in the cage bedding. The control group was unchanged in appearance. Animals killed at day 2 contained greatly distended ceca that were full of liquid and gas and had no solid stool in either proximal or distal portions of the colon. In addition, mice on the lactose diet lost approximately 7.0% 1.1% of their body weight during the diet period, a significant difference from control mice, which gained 1.8% 1.6% in weight (P 0.001). An estimate of the amount of fluid in the intestinal tract can be obtained by calculating the G/C ratio of each mouse. Animals fed the lactose-based diet had G/C ratios that were nearly twice that of control (0.133 vs ; P 0.001; Table 1), a figure exacerbated by, but not entirely due to, significant loss of body weight in these mice. By day 4, diarrhea in the animals in the lactose-fed group had resolved and no soft stool was observed on the cage bedding. Animals on the lactose diet, however, still displayed enlarged ceca that were gas and fluid filled. These mice also showed similarly increased G/C ratios when compared with control Table 1. G/C Ratios of Control and Experimental Mice After Two or Four Days on Study Day 2 G/C ratio SE Day 4 G/C ratio SE FVB/N control; n FVB/N lactose; n a a FVB/N PEG; n a ND GC-C / control; n ND GC-C / lactose; n b ND GC-C / PEG; n b ND GC-C / control; n ND GC-C / lactose; n c ND GC-C / PEG; n c ND NOTE. G/C ratios are presented as the mean SE. n, number of animals within a group. GC-C /, guanylate cyclase C heterozygote; GC-C /, guanylate cyclase C null; ND, not determined. a Significant difference from wild-type control (P 0.001). b Significant difference from GC-C / control group (P 0.001). c Significant difference from GC-C / control group (P 0.001). mice, although solid stool was evident in distal regions of the colon (0.123 vs ; P 0.001; Table 1). Northern Analysis of Intestinal NHE3 and DRA We initially wished to characterize this animal model of osmotic diarrhea by determining whether mice on the lactose diet were able to up-regulate absorptive pathways that are expected to increase in response to significant water loss. 29,32 As shown in Figure 1A, animals fed the lactose diet had increased levels of the NHE3 RNA in the proximal colon at days 2 and 4 when compared with control mice. The DRA gene recently has been shown to coordinate chloride and bicarbonate exchange and would be expected to be increased in this diarrheal model as well. 29 Figure 1B shows that RNA levels of DRA in the proximal colon are increased at day 2 of lactose feeding. By 4 days on the lactose diet, levels of DRA are more substantially induced when compared with control. These data suggest that, in mice, disaccharide-mediated malabsorption can cause diarrhea severe enough to initiate molecular mechanisms that attempt to decrease water and electrolyte loss. We next sought to determine the effect of lactose feeding on RNA levels of guanylin, uroguanylin, and their receptor, GC-C. Response of Guanylin, Uroguanylin, and GC-C to Osmotic Diarrhea Noting that the expected increases in absorptive pathways occurred in animals fed the lactose diet, we anticipated a decrease in RNA levels of prosecretory proteins such as guanylin and uroguanylin. Surprisingly, a striking and consistent increase in guanylin levels was

4 1194 STEINBRECHER ET AL. GASTROENTEROLOGY Vol. 121, No. 5 Figure 1. Colonic NHE3 and DRA mrna are increased after 2 or 4 days of lactose feeding. Northern analysis of proximal colon RNA from control and osmotic diarrhea mice was performed as stated in Materials and Methods. Graphical representations of (A) NHE3 and (B) DRA signal show increases after 2 or 4 days on the lactose diet. See text for normalization methods of Northern blotting signals for this and all figures. Data are presented as mean SE; asterisks show significance vs. control (P 0.05); n 4 animals per group. seen in most intestinal segments from mice fed the lactose diet for 48 hours. Specifically, an approximately 2-fold increase in guanylin RNA was apparent (Figure 2A and B). Only in the distal colon were levels of guanylin RNA not increased. Uroguanylin RNA levels (Figure 2C and D) were increased to an even greater degree than that of guanylin. Uroguanylin levels in the proximal jejunum were nearly 3-fold more than that of control levels, suggesting a robust response to the intraluminal lactose. Although cecal segments contained uroguanylin RNA levels that were double that of control, the large intestines of mice fed the lactose diet did not consistently show an increase in uroguanylin RNA. A 2-fold increase in uroguanylin RNA levels in the kidneys of mice on the lactose diet was also seen (Figure 2C and D). Having established that guanylin and uroguanylin RNA are increased after 2 days of lactose feeding, we next determined the effect of this diet on guanylin and uroguanylin RNA after 4 days. At this time point, the diarrhea seen in lactose-fed animals had resolved and solid stool was apparent in distal portions of the colon, suggesting that compensatory mechanisms were in place to either increase fluid absorption and/or decrease the osmotic load of the lactose in the intestine. Guanylin RNA expression had returned to that of control mice Figure 2. Guanylin and uroguanylin mrna is increased in animals with osmotic diarrhea. Twenty micrograms of total RNA from control mice and animals fed the lactose diet for 2 days were electrophoresed, blotted onto nylon membranes, and probed using a radiolabeled mouse guanylin or uroguanylin cdna probe. The signal was then quantitated and normalized against the 18S ribosomal subunit. All intestinal sections show an increase in (A) guanylin or (C) uroguanylin mrna levels with the exception of distal colon (n 6 12 per diet group). (B and D) Representative Northern blots show typical results from control and lactose-fed animals. Data are presented as mean SE; asterisks show significance vs. control (P 0.05); C, control diet; L, lactose diet; Gn, guanylin; Ugn, uroguanylin; 18S, 18S ribosomal subunit; PJ, proximal jejunum; IL, ileum; CEC, cecum; PC, proximal colon; DC, distal colon; Kid, kidney.

5 November 2001 GUANYLIN AND UROGUANYLIN IN OSMOTIC DIARRHEA 1195 Figure 3. Guanylin and uroguanylin mrna return to control levels as osmotic diarrhea resolves. Total RNA from control and mice on the lactose chow for 4 days was electrophoresed and blotted as described. Animals on the lactose diet did not have diarrhea by day 4 and solid stool was found in the distal colon on sacrifice. In lactose-fed animals, (A) guanylin and (C) uroguanylin levels had returned to near control with the exception of the proximal jejunal segments, which still show an increase (n 6 12 per diet group). (B and D) Representative Northern blots show typical results from control and lactose-fed animals. Data presented as mean SE; asterisks show significance vs. control (P 0.05); Con, control diet; Lact, lactose diet; Gn, guanylin; Ugn, uroguanylin; 18S, 18S ribosomal subunit; PJ, proximal jejunum; IL, ileum; CEC, cecum; PC, proximal colon; DC, distal colon. with the exception of a persistent increase in proximal jejunum (Figure 3A and B). Uroguanylin levels were increased in the small intestine of lactose-fed mice, but this difference was not significant compared with controls (Figure 3C and D). We next characterized GC-C RNA levels in the intestines of mice fed control or lactose diets. Unlike guanylin and uroguanylin, after 2 or 4 days on the lactose diet, there was no change in GC-C RNA in any segment of the intestine (data not shown). To determine levels of guanylin and uroguanylin prohormone in intestine, kidney, and serum of control and lactose-fed mice, we performed Western analysis using previously validated rat polyclonal antibodies, which recognize portions of mouse proguanylin (antibody 2538) and prouroguanylin (antibody 6910). 30,31 Figure 4A shows representative segments of the small and large intestine that were immunoblotted with proguanylinand prouroguanylin-specific antisera. Two days of lactose feeding and osmotic diarrhea resulted in substantial increases in prohormone levels throughout the small and large intestine of lactose-fed mice. Increases in the small intestine, as typified by proximal jejunal segments shown in Figure 4A, mirrored the up-regulation of RNA shown in Figure 2A D. Although the RNA response to the diarrhea was not significant in proximal colonic segments of lactose diet mice, proguanylin and prouroguanylin protein levels were greater in lactose-fed mice compared Figure 4. Intestinal proguanylin and prouroguanylin levels are increased by osmotic diarrhea. Western analysis was performed on 65 g of jejunal or colonic homogenate using proguanylin- and prouroguanylin-specific antibodies. (A) At 2 days on the lactose diet, consistent increases of both prohormones are seen in the jejunum and colon of mice on the lactose diet. (B) After 4 days of lactose feeding, proguanylin and prouroguanylin levels had returned to normal with the exception of cecum, in which they were still elevated. Con, control tissue; Lact, lactose diet tissue; Ab2538 Pro-Gn, antibody 2538 specific to proguanylin; Ab6910 Pro-Ugn, antibody 6910 specific to prouroguanylin.

6 1196 STEINBRECHER ET AL. GASTROENTEROLOGY Vol. 121, No. 5 with control animals (Figure 4A). Four days of lactose feeding resulted in prohormone levels that were unchanged from control with the exception of the cecum. Figure 4B shows that greater amounts of each prohormone in ceca of lactose diet mice continue to the 4-day time point. Up-regulation of uroguanylin RNA in the kidney of lactose-fed mice led us to determine prohormone levels in both kidney and sera of animals given the lactosebased diet. Increases were apparent in the kidney and serum of mice with osmotic diarrhea as exemplified by the typical blots shown in Figure 5. Increases in the level of renal prouroguanylin were apparent at day 2 in lactose-fed mice (Figure 5A). Prouroguanylin levels were typically greater after 2 days of osmotic diarrhea than that seen after 4 days (data not shown). Serum levels of prouroguanylin were also elevated after 2 days on the lactose diet (Figure 5B). These data suggest that this model of osmotic diarrhea is able to affect systemic regulation of prouroguanylin levels. Some receptors and their ligands coordinately control each other through regulatory feedback loops. 33,34 Therefore, to determine whether GC-C is necessary for the up-regulation of guanylin and uroguanylin in this model, we placed GC-C / and GC-C / animals on control and lactose-based diets for 48 hours and determined the RNA levels of each ligand. GC-C / and GC-C / mice that were placed on the lactose diet for 2 days did not respond with diarrhea or anogenital staining. This is caused by the C57BL/6 genetic background of these mice; subsequent studies have determined that wild-type C57BL/6 animals do not show outward signs Figure 5. Renal and serum levels of prouroguanylin are increased in lactose-fed mice. Western analysis of kidney homogenate and serum from mice on the lactose diet for 48 hours reveals an increase in prouroguanylin levels. Blots typical of several lactose diet studies show that (A) kidney prouroguanylin is substantially increased and (B) circulating levels of the prohormone are elevated above control. Ab6910 Pro-Ugn, antibody 6910 specific to prouroguanylin. of diarrhea like the FVB/N strain described above (data not shown). Despite this, G/C ratios in both GC-C / and GC-C / animals fed the lactose diet were increased in comparison to control animals of the same genotype (GC-C / vs , P 0.001; GC-C / vs , P 0.001; Table 1). G/C ratios in GC-C / mice were higher than GC-C / mice in all study groups (control, lactose, and PEG). The significance of these observations is uncertain. Total body weight loss was similar in GC-C / mice and GC-C / mice after 48 hours on the lactose diet. Increases in intestinal guanylin and uroguanylin were seen in GC-C / and GC-C / animals that were fed the lactose diet when compared with control animals of the same genotype. Although the magnitude of the guanylin and uroguanylin RNA response to lactose feeding was not of the same degree as that of wild-type FVB/N mice, guanylin and uroguanylin were increased in proximal jejunum of lactose-fed GC-C / mice (Figure 6A D). However, there was no increase in uroguanylin in the kidneys of GC-C / mice on the lactose diet when compared with control. This lack of response by renal uroguanylin to osmotic diarrhea occurred in wild-type C57BL/6 and GC-C / mice as well. Next, we determined the protein levels of prouroguanylin in GC-C deficient mice. When compared with control GC-C / animals, prouroguanylin levels in GC- C / mice on the lactose diet for 48 hours were increased to a similar degree as that seen in wild-type mice on the lactose diet (Figure 7A D). Prouroguanylin levels mirrored RNA increases in proximal jejunum (Figure 7A), whereas levels in proximal colon were increased despite the lack of a statistically significant rise in proximal colon RNA of lactose-fed mice (Figure 7B). No change was seen in prouroguanylin levels in the kidney (Figure 7C). Serum levels of prouroguanylin are elevated (Figure 7D), indicating that the systemic effects of this model occur independently of the presence of GC-C. Two days of lactose feeding also increases intestinal proguanylin levels in GC-C / mice to a degree similar to prouroguanylin increases shown in Figure 7 (data not shown). Dehydration and acute malnourishment that occur during the diet period could affect levels of both guanylin and uroguanylin. However, water deprivation for 24 or 48 hours did not alter guanylin or uroguanylin RNA levels in the intestine or uroguanylin levels in the kidney (data not shown). This suggests that the increases seen with the lactose diet are not solely because of its dehydrating effect. Similarly, when food was withheld from mice for 48 hours, there was no change in guanylin or

7 November 2001 GUANYLIN AND UROGUANYLIN IN OSMOTIC DIARRHEA 1197 Figure 6. Guanylin and uroguanylin RNA are increased in GC-C deficient mice. GC-C / mice were placed on control or lactose-rich diets and guanylin and uroguanylin RNA were measured. The absence of the receptor GC-C has no effect on guanylin and uroguanylin increases after 48 hours on the lactose diet. (A and B) Guanylin and (C and D) uroguanylin levels are increased significantly in proximal jejunum. See Results for further details. n 5 9 per diet group. Data are presented as mean SE; asterisks show significance vs. control (P 0.05); Con, control diet; Lact, lactose diet; Gn, guanylin; Ugn, uroguanylin; 18S, 18S ribosomal subunit; PJ, proximal jejunum; PC, proximal colon; Kid, kidney. uroguanylin RNA levels in the intestines of fasted mice when compared with control. Uroguanylin RNA was also unchanged vs. control levels in the kidneys of fasted mice. Western analysis of proguanylin and prouroguanylin levels in the intestine and kidney also indicated no appreciable change in mice that were dehydrated or acutely malnourished (data not shown). Effect of Hypertonic Water on Guanylin and Uroguanylin Levels in Wild-type FVB/N and GC-C Deficient Mice We have established that 48 hours of dehydration and fasting do not, but a lactose-based diet does, significantly influence guanylin and uroguanylin RNA and prohormone levels in the intestine and kidney. If the hypertonic nature of the lactose diet was the basis for guanylin/uroguanylin up-regulation, then another source of persistent luminal osmolytes would likely have the same effect. To determine whether another hypertonic dietary factor would cause guanylin and uroguanylin induction, we provided a 40 mmol/l PEG 3350 solution as the sole water source to wild-type FVB/N mice or GC-C / and GC-C / mice. Control mice of each strain and genotype received distilled water, and all groups had free access to standard mouse chow during the 48-hour study protocol. FVB/N mice that drank the PEG solution for 48 hours had liquid stool, anogenital staining, and elevated G/C ratios when compared with control mice (FVB/N PEG vs , P 0.001; Table 1). GC-C / and GC-C / mice responded to the PEG solution similarly, with liquid stool and increased G/C ratios (GC-C / PEG vs ; GC-C / PEG vs , P 0.001; Table 1). We determined the levels of guanylin and uroguanylin RNA in the proximal jejunum and proximal colon and uroguanylin RNA in the kidney of mice given PEG water. Both FVB/N (Figure 8) and GC-C / (Figure 9) animals responded to the PEG solution with elevated levels of guanylin and uroguanylin when compared with control mice of the same strain and genotype. Upon examination, we observed an approximate 2-fold increase in both guanylin and uroguanylin in the proximal jejunum of PEG-water mice vs. control (Figures 8 and 9). Mice with PEG-mediated diarrhea had variable and nonsignificant changes in levels of guanylin and uroguanylin in the proximal colon. Renal levels of uroguanylin RNA were greater in FVB/N mice given the PEG solution (Figure 8C and D). Although the lactose-based diet did not modulate renal uroguanylin levels in GC-C heterozygous or null mice, the PEG solution consistently elevated uroguanylin in the kidneys of GC-C deficient mice (and GC-C / mice; data not shown) vs. control animals (Figure 9C and D). The increases in intestinal guanylin and uroguanylin seen in GC-C / mice on the PEG solution were similar to the increases seen in GC-C /

8 1198 STEINBRECHER ET AL. GASTROENTEROLOGY Vol. 121, No. 5 Figure 7. In GC-C deficient mice, prouroguanylin is increased in the intestine and blood after 2 days on the lactose diet. Western analysis using prouroguanylin-specific antisera 6910 (Ab6910 Pro-Ugn) shows elevated levels of the prohormone in (A) proximal jejunum, (B) proximal colon, and (D) serum. (C) Kidney levels of prouroguanylin were not increased in GC-C / mice. mice with PEG solution (data not shown). Protein levels of proguanylin and prouroguanylin in FVB/N and GC- C / were increased in tissue as well as serum to a degree similar to that seen in the lactose-based osmotic diarrhea mice (data not shown). Discussion The commonly accepted role for guanylin and uroguanylin in the gastrointestinal tract is to induce secretion. These ligands activate GC-C and initiate an intracellular pathway that results in release of chloride and/or bicarbonate. It is thought that the extracellular ionic gradient that this establishes draws water across the cell layer. In this study, we asked whether, when confronted with an obligate loss of water, the intestine would respond by decreasing levels of guanylin and uroguanylin in an attempt to minimize fluid and electrolyte wasting. To answer this question, we established a mouse model of osmotic diarrhea by using the nonabsorbable osmolytes lactose or polyethylene glycol. This model is characterized by significant body weight loss, movement of water into the lumen of the intestine, and diarrhea. In addition, mrnas encoding NHE3 and DRA are greatly increased, suggesting that administration of these nonabsorbable factors sufficiently stresses the animal as to activate compensatory mechanisms that counteract water and electrolyte loss. There have been relatively few in vivo models of diarrhea in mice or rats that enable studies of physiologic regulation related to intestinal fluid loss. Previous use of magnesium citrate-phenolphthalein solutions or nonabsorbable factors alone have provided some data concerning the effects of diarrhea on the intestine in the rat Evidence suggests that rodents fed large amounts of lactose may rapidly increase lactase levels and begin to hydrolyze and absorb this disaccharide. 38,39 This may occur after 4 days on the lactose diet and, along with compensatory absorptive mechanisms, would explain the resolution of the diarrhea in this study. However, we detected no consistent increase in lactase levels after 4 days on the lactose diet (data not shown). We have used this animal model to address regulation of the putative secretagogues guanylin and uroguanylin in response to diarrhea and determined that these hormones are, unexpectedly, up-regulated. Two days on the lactose diet caused substantial increases in mrna levels of both genes in the intestine, and uroguanylin levels were also elevated in the kidney. In addition, protein levels of both prohormones were increased in the intestine and prouroguanylin was higher in the kidney and serum of lactose-fed animals. GC-C mrna was unchanged and the binding capacity and avidity of this receptor remained constant in this model of osmotic diarrhea (data not shown). Four days on the lactose diet resulted in both resolution of the diarrhea as well as the return of guanylin and uroguanylin mrna and prohormone to control levels in most segments of the intestine. In mice lacking GC-C, which is the only characterized receptor for guanylin and uroguanylin, the response to the luminal osmotic load was similar to wild-type animals. Both GC-C / and GC-C / animals had in-

9 November 2001 GUANYLIN AND UROGUANYLIN IN OSMOTIC DIARRHEA 1199 Figure 8. Guanylin and uroguanylin are increased in wild-type FVB/N after 48 hours with 40 mmol/l PEG drinking water. (A and B) Guanylin and (C and D) uroguanylin RNA were measured using Northern analysis and were increased after 48 hours of 40 mmol/l PEG solution in place of drinking water. Statistically significant increases in guanylin or uroguanylin RNA were seen in proximal jejunum and kidney. N 5 9 per group. Data are presented as mean SE; asterisks show significance vs. control (P 0.05). PJ, proximal jejunum; PC, proximal colon; Kid, kidney; Con, control; Gn, guanylin; Ugn, uroguanylin; 18S, 18S ribosomal subunit. creased levels of guanylin and uroguanylin in the proximal portions of the small intestine when fed the lactose diet but showed increases in renal levels of uroguanylin only when on the hypertonic PEG solution. Induction of renal uroguanylin was not seen in wild-type C57BL/6 mice on the lactose diet, indicating that genetic background is the likely cause of this lack of response. Strain variation may be expected because the compensatory physiologic response to the lactose diet is almost certainly multigenic and is likely influenced by genetic background. Unlike lactose, PEG is not easily digested by the intestine or fermented by intestinal flora and, Figure 9. Guanylin and uroguanylin are increased in GC-C / mice after 48 hours with 40 mmol/l PEG drinking water. (A and B) Guanylin and (C and D) uroguanylin RNA were measured using Northern analysis and were increased in proximal jejunum and kidney after 48 hours of 40 mmol/l PEG solution consumption. N 4 6 per group. Data are presented as mean SE; asterisks show significance vs. control (P 0.05). PJ, proximal jejunum; PC, proximal colon; Kid, kidney; con, control; Gn, guanylin; Ugn, uroguanylin; 185, 185 ribosomal subunit.

10 1200 STEINBRECHER ET AL. GASTROENTEROLOGY Vol. 121, No. 5 therefore, its ability to maintain a hypertonic environment in the intestinal lumen is greater than lactose and overwhelms the absorptive capacity of the intestine. This may explain the diarrhea seen in GC-C deficient mice on the PEG water solution that was absent in mice fed the lactose diet. Overall, the data presented here suggest that the increase in guanylin/uroguanylin seen in this model does not require the presence of GC-C and, therefore, it is unlikely that any regulatory cross-talk between ligand and receptor is necessary to respond to acute luminal hypertonicity. Considering the prevailing evidence for guanylin and uroguanylin as intestinal secretagogues, up-regulation of guanylin and uroguanylin in response to osmotic diarrhea is unexpected. The increase in extraintestinal prouroguanylin is especially surprising because the diuretic and natriuretic effects of circulating uroguanylin would be expected to exacerbate the dehydration inherent to conditions of osmotic diarrhea. 17,18 Thus, the potential significance of up-regulation of uroguanylin in the kidney is uncertain. It should be noted that increased systemic levels of prouroguanylin may be the result of several factors, including a change in prohormone processing, a higher rate of prohormone release from intestine or kidney, and/or a slower rate of clearance from the blood, all possibilities that require further investigation. The effects of this diarrhea model are specific with respect to guanylin/uroguanylin because fasting and dehydration do not lead to increases in their RNA or protein levels, suggesting that these secondary effects of osmotic diarrhea are not the cause of guanylin/uroguanylin induction. Both the lactose diet as well as the PEG drinking water had similar positive effects on guanylin and uroguanylin expression. Taken collectively, these findings are consistent with the interpretation that these genes respond to the intraluminal hypertonicity that is inherent in this model. Hypertonicity has been shown previously to increase secretion of guanylin and uroguanylin. Kita et al. have shown greater release of guanylin and, to a lesser degree, uroguanylin, in response to a short term (1 hour) intraluminal infusion of hypertonic fluid in the small intestine. 40 A major effect of the lactose and PEG diets is to create a very strong osmotic gradient in the intestine. In this respect, the impetus for the increase in guanylin and uroguanylin secretion seen by Kita et al. and the up-regulation of these ligands shown in this study are similar. Hypertonicity-induced release of guanylin and uroguanylin that results in fluid secretion and dilution of the luminal contents has been suggested by Kita et al. 40 It is possible that one role of guanylin/uroguanylin may be to diminish the osmotic stress experienced by intestinal epithelia upon ingestion of hypertonic meals. Although this hypothesis seems consistent with this and other studies, it is counterintuitive that the intestine would require a secretory pathway to aid in transferring water into the gut lumen because fluid is thought to flow freely across the leaky epithelia of the small intestine in response to luminal hypertonicity. Furthermore, G/C ratios are increased in GC-C / mice given hypertonic diets compared with controls (Table 1), suggesting a limited role for the (uro)guanylin/gc-c pathway in hypertonicity-induced luminal content dilution. The hydration state of the intestinal lumen could play a role in post-release regulation of guanylin/uroguanylin activity. Intraluminal fluid levels may influence the concentration and, therefore, the activity of these ligands and this may explain the lack of down-regulation in response to osmotic diarrhea. If guanylin and uroguanylin do act as intraluminal fluid sensors that are essentially down-regulated by dilution during osmotic diarrhea, then the absence of a transcriptional and translational decrease of guanylin/uroguanylin is not surprising. Control of (uro)guanylin activity through luminal hydration status does not, however, explain the up-regulation of these ligands presented here. Further studies to determine guanylin/uroguanylin levels in response to other intestinal hydration states are in order. A significant function of proguanylin/prouroguanylin, or their active peptides, may be distinct from the characterized secretory pathway. It is possible that biological activity is present in the prohormones before or after cleavage of the GC-C binding guanylin and uroguanylin polypeptides. Evidence is consistent with the possibility that there is another receptor for the STa-guanylinuroguanylin ligand family separate from GC-C. 18,41 Although these possibilities are largely speculative at this time, the existence of an alternative function for the GC-C receptor and/or the prohormones is consistent with the up-regulation of guanylin and uroguanylin demonstrated in this model of osmotic diarrhea. References 1. Vaandrager AB, Bot AG, Ruth P, Pfeifer A, Hofmann F, De Jonge HR. Differential role of cyclic GMP-dependent protein kinase II in ion transport in murine small intestine and colon. Gastroenterology 2000;118: Vaandrager AB, Smolenski A, Tilly BC, Houtsmuller AB, Ehlert EM, Bot AG, Edixhoven M, Boomaars WE, Lohmann SM, de Jonge HR. Membrane targeting of cgmp-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl channel activation. Proc Natl Acad Sci USA1998;95:

11 November 2001 GUANYLIN AND UROGUANYLIN IN OSMOTIC DIARRHEA Vaandrager AB, Edixhoven M, Bot AGM, Kroos MA, Jarchau T, Lohmann S, Genieser HG, de Jonge HR. Endogenous type II cgmp-dependent protein kinase exists as a dimer in membranes and can be functionally distinguished from the type I isoforms. J Biol Chem 1997;272: Chabot H, Vives MF, Dagenais A, Grygorczyk C, Berthiaume Y Grygorczyk R. Down-regulation of epithelial sodium channel (ENaC) by CFTR co-expressed in Xenopus oocytes is independent of Cl conductance. J Membr Biol 1999;169: Kunzelmann K, Kiser GL, Schreiber R, Riordan JR. Inhibition of epithelial Na currents by intracellular domains of the cystic fibrosis transmembrane conductance regulator. FEBS Lett 1997; 400: Field M, Graf LH Jr, Laird WJ, Smith PL. Heat-stable enterotoxin of Escherichia coli: in vitro effects on guanylate cyclase activity, cyclic GMP concentration, and ion transport in small intestine. 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Dietary regulation of intestinal lactase and sucrase in adult rats: quantitative comparison

12 1202 STEINBRECHER ET AL. GASTROENTEROLOGY Vol. 121, No. 5 of effect of lactose and sucrose. J Pediatr Gastroenterol Nutr 1985;4: Goda T, Bustamante S, Thornburg W, Koldovsky O. Dietary-induced increase in lactase activity and in immunoreactive lactase in adult rat jejunum. Biochem J 1984;221: Kita T, Kitamura K, Sakata J, Eto T. Marked increase of guanylin secretion in response to salt loading in the rat small intestine. Am J Physiol 1999;277:G960 G Mann EA, Cohen MB, Giannella RA. Comparison of receptors for Escherichia coli heat-stable enterotoxin: novel receptor present in IEC-6 cells. Am J Physiol 1993;264:G172 G178. Received February 6, Accepted July 12, Address requests for reprints to: Mitchell B. Cohen, M.D., Division of Pediatric Gastroenterology and Nutrition, Children s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio mitchell.cohen@chmcc.org; fax: (513) Supported by a grant from the National Institutes of Health, DK The authors thank Drs. Manoocher Soleimani and Hassane Amlal for critical input and James Knapmeyer and Jennifer Hawkins for technical assistance.