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ANIMAL NUTRITION |
,3
Department of Animal Science, Cornell University, Ithaca, NY 14853 and
and
Renal Division, Emory University, Atlanta, GA 30322
Abstract
Urea recycling in ruminants has been studied extensively in the past, but the mechanisms regulating the amount of urea recycled or excreted remain obscure. To elucidate the role of urea transporters (UT) in N recycling, nine Dorset-Finn ewe lambs (20.8 ± 0.8 kg) were fed diets containing 15.5, 28.4, and 41.3 g of N/kg of DM for 25 d. Nitrogen balance and urea N kinetics were measured during the last 3 d of the period. Animals were then slaughtered and mucosa samples from the rumen, duodenum, ileum, and cecum, as well as kidney medulla and liver, were collected. Increasing N intake tended to increase N balance quadratically (1.5, 5.1, and 4.4 ± 0.86 g of N/d, P < 0.09), and linearly increased urinary N excretion (2.4, 10, and 16.5 ± 0.86 g N/d, P < 0.001) and plasma urea N concentration (4.3, 20.3, and 28.4 ± 2.62 mg of urea N/dL, P < 0.001), but did not affect fecal N excretion (5.0 ± 0.5 g of N/d; P < 0.94). Urea N production (2.4, 11.8, and 19.2 ± 0.83 g of N/d; P < 0.001) and urinary urea N excretion (0.7, 7.0, and 13.4 ± 0.73 g N/d; P < 0.001) increased linearly with N intake, as well as with the urea N recycled to the gastrointestinal tract (1.8, 4.8, and 5.8 ± 0.40 g of N/d, P < 0.001). No changes due to N intake were observed for creatinine excretion (518 ± 82.4 mg/d; P < 0.69) and clearance (46 ± 10.7 mL/min; P < 0.56), but urea N clearance increased linearly with N intake (14.9, 24.4, and 34.9 ± 5.9 mL/min; P < 0.04). Urea N reabsorption by the kidney tended to decrease (66.3, 38.5, 29.1 ± 12.6%; P < 0.06) with increasing N content of the diet. Increasing the level of N intake increased linearly the weight of the liver as a proportion of BW (1.73, 1.88, and 2.22 ± 0.15%, P < 0.03) but only tended to increase the weight of the kidneys (0.36, 0.37, and 0.50 ± 0.05%, P < 0.08). Urea transporter B was present in all the tissues analyzed, but UT-A was detected only in kidney medulla, liver, and duodenum. Among animals on the three diets, no differences (P > 0.10) in UT abundance, quantified by densitometry, were found. Ruminal-wall urease activity decreased linearly (P < 0.02) with increasing level of N intake. Urease activity in duodenal, ileal, and cecal mucosa did not differ from zero (P > 0.10) in lambs on the high-protein diet. In the present experiment, urea transporter abundance in the kidney medulla and the gastrointestinal tract did not reflect the increase in urea-N reabsorption by the kidney and transferred into the gut.
Key Words: Biochemical Transporters • Kinetics • Nitrogen Metabolism • Sheep • Urea Recycling
Introduction
It is not uncommon for ruminants to synthesize urea N in excess of the N apparently digested (Lobley and Lapierre, 2001
), implicating that for these animals to be in a positive N balance, a portion of the urea synthesized has to recycle into the gastrointestinal (GI) tract and back into the ornithine cycle. Nitrogen recycled into the GI tract can then be used for microbial protein synthesis in the rumen and thus provide AA to the host. Ruminants on low-protein diets depend more on their ability to recycle urea N than animals fed adequate levels of protein (Siddons et al., 1985; Marini and Van Amburgh, 2003). The role of the kidney in the salvage of urea N has been well established (Faix et al., 1988; Tebot et al., 1998; Cirio et al., 1990), but it is not clear how ruminants can achieve higher clearances of urea N into the GI tract when fed low-protein diets (Ford and Milligan, 1970; Kennedy and Milligan, 1980; Marini and Van Amburgh, 2003). The discovery of urea transporters (UT; You et al., 1993), their role in animals fed low-protein diets (Isozaki et al., 1994), and their identification in the GI tract of ruminants (Ritzhaupt et al., 1997; 1998) provide a potential mechanism by which the transfer of urea N into the GI tract could be regulated. Previous work (Marini and Van Amburgh, 2003) showed that protein intake might induce a differential abundance of UT in the ruminal mucosa of Holstein heifers. The objective of the present work was to further elucidate the role of UT in gut tissue, liver, and kidney, and their relationship with urea N recycling in lambs fed different protein levels.
Materials and Methods
Animals and Diets
Nine Dorset-Finn ewe lambs (20.8 ± 0.64 kg) were used in a completely randomized design to investigate the effect of N intake on N kinetics and UT abundance. Care, handling, and sampling of the lambs were in accordance with the guidelines issued by the Cornell University Animal Care and Use Committee. The experimental protocol was approved by the Cornell University Institutional Animal Care and Use Committee (Protocol No. 01-36). Lambs were housed at the Large Animal Research and Teaching Unit in individual metabolism cages for 10 d and then transferred to individual stalls for 2 wk in a temperature-controlled environment (18 to 20°C) under continuous lighting and background noise.
Ewe lambs were fed individually 71 g of DM/kg BW0.75 daily of a diet comprised of 15% brome hay and 85% of a pelleted feed twice daily at 0800 and 2000. Dietary ingredients have been described elsewhere (Marini and Van Amburgh, 2003). In brief, there were two pellets, one for Diet 1 (D1, low-protein pellet, 9.8% CP) and the other for Diet 3 (D3, high-protein pellet, 28.1% CP), and by mixing them in equal parts, ingredients for D2 were obtained. The actual N content of the diets was 15.6, 28.7, and 40.5 g of N/kg of DM. Diets were formulated to be isocaloric on a ME (2.75 Mcal/kg of DM) basis and to contain similar levels of NDF (Marini and Van Amburgh, 2003). Orts were collected once a day and stored until the end of the sampling period, when they were weighed, pooled, and sampled for analysis. The experimental period lasted 25 d and included a 21-d adaptation to the diet, a 3-d total collection of fecal and urinary excretions, and a final day when animals were slaughtered and tissue collected. Animals had access to water at all times. Lambs were weighed weekly without feed or water restriction.
Sample Collection
At feeding time (0800), and 3, 6, and 9 h later on d 22, blood was collected from the jugular vein by venipuncture into Vacutainers containing heparin (Becton Dickinson, Rutherford, NJ). Blood samples were placed on ice and centrifuged at 1,500 x g for 15 min and the plasma obtained stored at -20°C.
On d 22, immediately after the 0800 bleeding, a single dose of 1.6 mmol of 15N15N-urea (Mass Trace, Woburn, MA) in 20 mL of sterile saline (9 g of NaCl/L) was injected intravenously and urine was collected for the following 72 h.
Feces and urine were collected daily between d 23 and 25 before the 0800 feeding and weighed to determine output; a 10% subsample of urine and a 25% subsample of feces were collected, composited by period, and stored at -20°C until later analysis. Urine was collected from the bladder with an 8-gauge Foley catheter (Bard Medical Division, Covington, GA) into a plastic container with enough 50% H2SO4 to reduce pH below 2.5. Initially, urine and fecal collection was planned for 5 d, but was reduced to a 3-d collection period because the Foley catheters produced bladder irritation and discomfort.
After completing the N balance study, the ewe lambs were slaughtered in the abattoir of the Animal Science Department. Animals were stunned with a captive bolt and killed by exsanguination. Gastrointestinal tissue samples were taken from the ventral sac of the rumen, proximal duodenum, terminal ileum, and the blind sac of the cecum and rinsed with ice-cold saline solution (NaCl 9 g/L). Rumen mucosa was stripped from the muscular layers, and the serosa and muscular layers of the intestine were carefully removed, leaving mostly the mucosa layer. Samples were then taken with a sterile 6-mm biopsy punch (Miltex Instrument Co., Inc., Bethpage, NY) from areas where only mucosa was present, placed in a 2.0-mL Eppendorf tube, immediately frozen in liquid N, and stored at -80°C until determination of urease activity or UT. A sample from the left lobe of the liver was collected, as well as from the kidney medulla, and both samples were frozen in liquid N and stored at -80°C until UT analysis.
Sample Analysis
Urinary urea was isolated using a cation exchange resin (AG 50W-X8, 100 to 200 mesh hydrogen form, Bio-Rad, Richmond, CA), its concentration determined by the diacetyl monoxime method of Marsh et al. (1957) using a Technicon autoanalyzer (Technicon Instruments Corp., Tarrytown, NY), and diluted to a final concentration of 6 mmol/L. The solution was then reacted under vacuum with lithium hypobromite, leading to a monomolecular degradation of urea into N2 (Walser, 1954). Details of this procedure have been discussed elsewhere (Marini and Van Amburgh, 2003). The N2 obtained was read with a NC2500 Carlo Erba dual inlet elemental analyzer (Thermoquest, Milan, Italy) as m/z 28, 29, and 30 corresponding to the 14N14N, 14N15N, and 15N15N parent urea molecule, respectively. The model of Lobley et al. (2000) was used to calculate urea N kinetics.
Plasma urea N (PUN) and urinary urea N (UUN) were determined by the diacetyl monoxime method as described previously. Feed, orts, fecal (fresh feces), and urinary N were measured by macro Kjeldahl (AOAC, 1990) that was modified to include the use of boric acid and steam distillation (Pierce and Haenisch, 1940). Plasma and urine creatinine were determined by the Jaffe reaction with a commercial kit (Sigma 555-A, Sigma, St. Louis, MO).
Urease activity was determined by incubating punched tissue samples from ruminal, duodenal, ileal and cecal mucosa in a PBS-urea solution (1 mL, pH = 7.4, ionic strength 10 mmol/L, 16 mmol of urea N/L) for 60 min at room temperature, vortexing frequently to allow adequate mixing between the tissue and the solution. Blanks (without tissue) were run alongside the samples and used as controls. The urea that disappeared during the incubation was assumed to have been hydrolyzed by bacterial urease present in the tissue. The reaction was terminated by the addition of 20 µL of 50% H2SO4 (vol/vol). The urea remaining in the eppendorf tubes was measured by the method of Marsh et al. (1965) as described above.
Liver and kidney tissue samples, as well as ruminal, duodenal, ileal and cecal mucosa, were homogenized in isolation buffer (10 mM triethanolamine, 250 mM sucrose, 1 µg/mL leupeptin, 0.1 mg/mL phenylmethylsulfonyl fluoride, pH 7.6, 0.025 to 0.1 g of tissue/mL of isolation buffer) for the determination of UT. Concentrated SDS was added to achieve a final concentration of 1%, then samples were sheared by passage through a 28-gauge needle and centrifuged for 15 min at 14,000 x g. Protein concentration was determined using the BioRad DC protein assay kit (BioRad, Richmond, CA). Proteins (10 µg/lane) were separated on 10% SDS-polyacrylamide gels, and then transferred to a polyvinylidene difluoride membrane (Gelman Scientific, Ann Arbor, MI), and Western blot analysis was performed as described previously (Kim et al., 2003). Membranes were probed with two antibodies as follows: 1) affinity-purified polyclonal antibody to UT-A (3.8 mg/mL used at 1:5,000 for Western blot). This antibody was prepared against the C-terminal portion of UT-A1 and will also detect UT-A2 and UT-A4 (Naruse et al., 1997); or 2) affinity purified polyclonal anti-UT-B (0.94 mg/mL diluted 1:3,000 for Western blot). The UT-B antibody was prepared against the carboxyl-terminal 19 AA of human UT-B and was able to recognize UT-B protein in cultured bovine aortic endothelial cells (Timmer et al., 2001). We previously confirmed the specificity of protein recognition by these antibodies by probing with preimmune serum and by performing peptide competition studies (Naruse et al., 1997, Timmer et al., 2001). This was reconfirmed in the present study by performing peptide competition studies for each antibody in each tissue (data not shown).
The antibodies were diluted in Tris-buffered saline (TBS) with 0.5% Tween-20. Blots were incubated with the primary antibody overnight at 4°C and then washed three times in TBS/Tween. Blots were then incubated with horseradish peroxidase-linked goat anti-rabbit IgG diluted 1:5,000 (Amersham, Arlington Heights, IL) for 2 h, at room temperature, and washed twice with TBS/Tween. The immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL). Autoradiograms were scanned using the Bio-Rad Gel Doc 1000 digital imaging densitometer. Scanned bands were quantified using the system’s Multi-Analyst version 1.0.1 software.
Where multiple bands were observed resulting from multiple forms of a single protein (UT-A1: 110–120 kDa; UT-B: 41–54 kDa), all bands in the group were measured together and designated using the molecular mass of the major form. In all cases, parallel gels were stained with Coomassie blue to confirm uniformity of loading (data not shown). Results are expressed as arbitrary units per microgram of protein.
Statistical Analysis.
Data were analyzed using the GLM procedure of SAS (Windows Version Release 8.02, SAS Inst., Inc., Cary, NC). The model used was Yij = µ + TRTi + eij, where Yij was the j observation for the dietary treatment TRTi (15.6, 28.7, or 40.5 g N/kg DM), eij the error associated with that observation, and µ the overall mean. Linear and quadratic contrasts were performed and the overall means obtained were assessed for significant differences at P < 0.05.
Results
The initial experimental diet included 30% brome hay, identical to a diet used in a previous experiment with Holstein heifers (Marini and Van Amburgh, 2003). This diet was fed to a group of lambs not used in the current experiment and found that to avoid orts, the proportion of hay had to be reduced to 15%. Despite the reduction in hay content, lambs left some feed (mainly hay) uneaten during the experiment. The actual DM and N intakes of the ewe lambs in the experimental diets are found in Table 1. Because animals on Diet 2 were slightly heavier and had almost no orts, a quadratic effect on N intake was detected. Increasing N intake linearly increased the N excreted in the urine (P < 0.001) but had no effect on the N excreted in the feces (P < 0.94; Table 1). Despite the large numerical difference observed in N balance, only a quadratic tendency (P < 0.09) was observed due to variation encountered which may have been associated with the small number of animals used (Table 1). Likewise, the observed weight gain showed a quadratic tendency (P < 0.09) and was consistent with the N balance data.
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). When expressed in relationship to the N ingested, urea production accounted for almost 75% of the N ingested in animals on Diet 3 compared with 58 and 27% in animals on Diets 2 and 1, respectively (P < 0.001; Table 1). Despite a narrow range (20 to 24%) in the proportion of recycled urea N/ N intake, we observed a quadratic effect (P < 0.05). Urea N excretion represented 7.6, 34.4, and 52.1% of the N intake for animals on Diets 1, 2, and 3, respectively (P < 0.001).
Increasing N intake had no effect on plasma creatinine concentration (P < 0.44; Table 2), nor on urinary excretion of creatinine (P < 0.68). Creatinine clearance remained constant (P < 0.56) among diets, but urea N clearance increased linearly (P < 0.04) with increasing levels of N fed. The consequences of increasing the level of N intake were an increase in the renal tubular load of urea N (P < 0.004) and urea N excretion (P < 0.001) accompanied by a tendency (P < 0.06) in the reduction of urea N reabsorbed by the kidney. The clearance of urea N into the GI tract decreased linearly (P < 0.02) with increasing level of dietary N (Table 2).
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. Increasing N intake decreased linearly ruminal (P < 0.02), duodenal (P < 0.04), ileal (P < 0.002), and cecal (P < 0.07) urease activity. The urease activity of the duodenal, ileal and cecal mucosa of the lambs on D3 was not different from zero (P > 0.10).
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) as a proportion of BW. Urea transporter B was present in all the tissues analyzed, but UT-A was only present in kidney medulla, liver and duodenum. Among animals on the three diets, no differences (P > 0.10) in UT abundance in any of the tissues analyzed as quantified by densitometry were found (Table 5).
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). For UT-B, rumen mucosa presented multiple bands. The smear between 40 and 50 kD is consistent with the size of UT-B in rat red blood cells, and the smear between 40 and 50 kD and the 98 kD band are consistent with the bands detected in kidney (Timmer et al., 2001). The kidney medulla (Figure 2) showed well-defined bands at 98, 40 to 55, and 36 kD for UT-B, but faint bands at 98 to 117 and 45 to 55 kD for UT-A. Liver (Figure 3) showed a single band at 45 kD for UT-A and two bands (45 and 98 kD) for UT-B.
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The adaptive changes to a low-N diet observed in the present experiment were consistent with the changes reported in the literature. Ruminants fed diets low in N decreased the urinary excretion of N and urea N, whereas urea N as a proportion of the total N excreted decreased (Ergene and Pickering, 1978; Leng et al., 1985; Tebot et al., 1998). Usually, if feed intake is not depressed, fecal N remains similar regardless of the N content of the diet (Siddons et al., 1985; Marini and Van Amburgh, 2003). Due to the number of animals used in this experiment, only a trend in N retention and weight gain was observed.
Consistent with the changes in urea N reabsorption reported in the literature (Tebot et al., 1985), the renal tubular load of urea N (due to a lower PUN) decreased, but the proportion of urea N reabsorbed tended to increase with decreasing levels of N fed. Some authors have reported a reduction in the renal plasma flow and glomerular filtration rate of sheep on a reduced N intake (Ergene and Pickering, 1978; Tebot et al., 1985), but we were not able to detect any difference in the clearance of creatinine among the diets. Schmidt-Nielsen and coworkers (Schmidt-Nielsen and Osaki, 1958; Schmidt-Nielsen et al., 1958) were the first to suggest that the kidney tubules were involved in the reabsorption of urea and that animals were able to increase urea reabsorption when fed low N diets. The increase in urea reabsorption by the kidney of rats fed low-N diets has been characterized (Isozaki et al., 1994), providing a mechanism of action for the increase in urea reabsorption observed by Schmidt-Nielsen and coauthors. The increase in urea N reabsorption by the kidney in the present experiment could not be explained by an increase in the abundance of UT in the renal medulla. However, the present studies do not address whether there is an increase in the function (Isozaki et al., 1994) or phosphorylation (Zhang et al., 2002) of UT-A1 that could explain the increase in urea N reabsorption. In addition, it has to be noted that although a higher proportion of urea-N was reabsorbed in Diet 1, a larger total amount was reabsorbed in Diets 2 and 3.
Multiple protein bands are detected for both UT-A and UT-B (reviewed in Sands, 2003; Bagnasco, 2003). However, the functional differences, if any, between these different proteins is not understood at present. Although UT-A protein expression was initially thought to be limited to the kidney, we showed that liver expresses 49- and 36-kD UT-A proteins, and that the abundance of the 49-kD liver UT-A increases in rats made uremic by nephrectomy, consistent with an increase in the activity of the ornithine cycle and the need to increase the efflux of urea from the hepatocytes (Klein et al., 1999). The same condition arises when animals are fed high-N diets. In the present experiment, lambs on Diet 3 produced eight times more urea N than animals on Diet 1, but we were unable to detect any changes in the hepatic UT-A and UT-B abundance among the different treatments. The increase in the relative weight of the liver is consistent with previous reports in sheep (Wester et al., 1995; McNeill et al., 1997), and the increase in the kidney weight of the lambs on the high-N diet was similar to the hypertrophy observed in mice fed high-N diets (Schrijvers et al., 2002). The increased weight of the heart with N restriction is consistent with previous reports in sheep (Wester et al., 1995; McNeill et al., 1997). The changes observed in heart weight were probably due to hypertrophy (Pissaia et al., 1980; Schweistahl et al., 1982), which results from the high level of catecholamines present (not measured in the present study) in the N-restricted animals (Pissaia et al., 1980).
The increase in the clearance of urea N into the GI tract was not accompanied by changes in the abundance of UT in the rumen, duodenum, ileum, or cecum. In a previous experiment with Holstein heifers (Marini and Van Amburgh, 2003) fed similar diets, we observed a sevenfold increase in the GI tract urea N clearance between the lowest and highest level of N fed. In the present experiment, there was only a twofold increase, probably because the lambs were able to maintain higher PUN levels than were the heifers. When sheep and cattle are fed identical diets (Thornton, 1970; Griffin et al., 1993), sheep generally have higher PUN due to the higher reabsorption of urea N by the kidneys (Thornton, 1970).
The urease activity of bacteria attached to the mucosa has been proposed as the mechanism controlling the passage of urea into the rumen (Cheng and Wallace, 1979; Wallace et al., 1979). We observed that the attached microbial population of lambs fed Diet 1 had higher urease activity than that of animals on the higher N diets. Because we only determined the enzymatic activity of the attached bacteria, the total urease activity of ruminal contents could not be estimated. The approximately 70% decrease in urease activity between Diet 1 and Diet 3 falls between the mild inhibition (30%) reported by Bunting et al. (1989) and the fivefold reduction reported by Cheng and Wallace (1979). But, even the low ruminal urease activities reported by these authors in sheep fed high-N diets were enough to hydrolyze approximately 10 times the urea N recycled to the total GI tract of lambs on Diet 3 in this experiment. Therefore, it seems unlikely that urease activity is an important regulator of the transfer of urea into the GI tract (Norton et al., 1982).
Urea is highly hydrosoluble and the urea permeability of cells is 100- to 1,000-fold greater than that of lipid bilayers, suggesting that proteins are involved in the transport of this molecule (Ripoche and Rousselet, 1996). Although we were unable to relate the presence of known UT to the urea clearance by the kidney and the GI tract, some other UT that are not as well characterized are still potential candidates for the transfer of urea across the gut mucosa. Active urea transporters have been described in the kidney of rats fed low-N diets (Isozaki et al., 1994), and active processes have been involved in the transfer of urea across the ruminal wall (Mooney and O’Donovan, 1970). More research, with a larger number of animals, is needed to confirm the present results and to further elucidate the mechanisms by which ruminants are able to redirect urea into the GI tract instead of excreting it in the urine.
Implications
The physiological adaptations to low-protein diets include increased reabsorption and salvage of urea by the kidney and an increase in the clearance of urea into the gastrointestinal tract. Although urea transporters are a promising candidate mechanism for the regulation of these events, we were unable to relate the abundance of known urea transporters to the urea transactions in ewe lambs fed different protein levels.
Footnotes
1 Supported by the Cornell Univ. Agric. Exp. Stn. 
2 We thank L. Lintault for her assistance with sample collection and analysis.
3 Correspondence and present address: 432 Animal Sciences Laboratory, University of Illinois, Urbana 61801 (phone: 217-244-2870; fax: 217-333-8804; e-mail: jcmarini{at}uiuc.edu).
Received for publication March 18, 2003. Accepted for publication November 10, 2003.
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