The rapid development of precocial goats in the first weeks after birth requires an adequate adaptation of phosphate transport systems to maintain the P homeostasis at each developmental stage. Here we examined the age-related development of Na+-Pitransport systems in small intestines, kidneys, and parotid glands of goats. Kinetic parameters were determined by brush-border membrane vesicle uptake studies, and relative expression of NaPi type II mRNA and protein was recorded by molecular biological methods. High intestinal Pi transport capacity was already present on the first day of life. Within the first 3 wk of life there seemed to be a change in the type of Na+-dependent Pitransporter, and NaPi IIb was expressed increasingly up to the fifth month of life. Renal Na+-Pi transport capacity was also high at birth, and this was associated with high expression levels of NaPi IIa mRNA, indicating the important role of this transporter for renal Pi reabsorption. At weaning an increase in both intestinal and renal Na+-Pitransport balanced the increasing requirements for Pi to establish the endogenous Pi cycle. Salivary Piconcentration and parotid NaPi II mRNA rose markedly to guarantee an adequate Pi supply for rumen microbes. We concluded that the high demand for Pi in young goats was assured by high basal Na+-Pi transport capacity of small intestines and kidney expressed continuously during ontogenesis.
- ontogenesis of sodium-phosphate transport
- small intestine
- parotid gland
- NaPi type II cotransporters
developing young organisms have a high requirement for phosphorus (P) for the mineralization of the skeleton and maintenance of metabolism and growth. Newborn precocial species such as goats are able to stand up within a few hours after birth, which implies a well-developed bone mineralization and an adequate energy metabolism for mobility and thermoregulation. In contrast, altricial species such as mice or rabbits do not have a comparable demand for P due to their stage of maturation at birth. To meet the high P requirement of developing goats, there must be adequate intestinal Pi absorption and renal Pi reabsorption as well. In adult goats epithelial Pi transporters in the apical membranes in the small intestines and in the kidney are characterized structurally and functionally. In the jejunum, a Na+-dependent Pi transport has been characterized that was molecularly based on a NaPi type IIb cotransporter (9, 14). In the kidney cortex, Pi reabsorption is mediated by a NaPi type IIa cotransporter, but its quantitative proportion on overall renal Na+-dependent Pi transport is still unknown (15). When young preruminant goats are gradually adapted to a solid diet, the endogenous Pi cycle will start to develop because of the increasing ability of the salivary glands, particularly the parotid glands, to transport Pi from blood to saliva. There is some experimental evidence that Pitransport in the salivary glands is Na+ dependent and basolateral localized (19), but the exact cellular mechanisms and localization of transporters are not yet fully understood. Salivary secretion is stimulated by mechanical as well as chemical stimuli of mouth and rumen mucosa receptors when intake of dietary roughage begins.
When the endogenous Pi cycling is established, Pi from intestinal absorption and renal reabsorption is recycled by concentrating Pi in the saliva and secreting it with the saliva into the rumen. Thus, at a continuous salivary secretion rate of 6–16 l/day in adult small ruminants with a mean salivary Pi concentration of 25 mmol/l, 2.4–6.4 g P/day flows into the rumen. High salivary concentrations of both Pi and bicarbonate prevent ruminal acidification by short-chain fatty acids (SCFA), which are the major end products of microbial fermentation. Furthermore, Pi is necessary to build up microbial cell walls, to form energy-rich phosphates, and to synthesize DNA for microbial growth and metabolism (7). Thus the development of the endogenous Pi cycle can be considered a strategy to ensure rumen microbial homeostasis to provide adequate amounts of SCFA as energy sources and high-quality microbial protein to the host organism.
There must be a sufficient postnatal development of Pitransport systems to guarantee an adequate Pi homeostasis at each stage of the developmental process. As this is poorly understood, it was the aim of this study to characterize the ontogenesis of the epithelial Pi transport systems in goats. The age groups studied spanned four stages of development: suckling kids in the 1st week of life, suckling kids in the 4th to 5th week of life, weaning kids in the 8th to 11th week of life, and ruminating goats in the 4th to 5th month of life. This corresponds to the four food intake periods: colostrum/first milk, pure milk, milk and first crude fiber, and milk-free, adult food. Expression of Pi transporters was examined in the jejunum, the kidney, and the salivary gland at the molecular and functional levels. For the first time, a NaPi type II transporter was demonstrated in the parotid glands.
MATERIALS AND METHODS
Animals and Feeding
Male kids of the Saanen-type breed Weisse Deutsche Edelziege were used for the experiments. The kids were between 1 and 150 days of age (see Table 1 for detailed information on groups, age, feeding and housing, and body weights). Plasma and saliva samples were taken to determine P status of the kids in each age group. Saliva was collected by placing a sponge close to the orifice of the parotid duct for a few minutes according to Boehnke et al. (3). General care and handling of the animals were approved and supervised by the Animal Welfare Commissioners of the University of Bonn and the School of Veterinary Medicine Hannover in accordance with the German Animal Welfare Law.
Animals were killed by stunning using a captive bolt pistol and bled from the carotid arteries. Kidneys, parotid glands, and midjejunal segments were obtained within 3–5 min after slaughter. After rinsing with ice-cold saline (0.9% NaCl wt/vol), parotid glands, jejunal mucosa, and kidney cortexes were frozen in liquid nitrogen and stored at −80°C for structural and functional analyses.
NaPi II-Specific RT-PCR in the Parotid Gland
RT-PCR was performed using poly(A)+ RNA isolated from parotid glands. Using 500 U Moloney mouse leukemia virus (MMuLV) RT (MBI Fermentas, Vilnius, Lithuania), 5 μg poly(A)+ RNA was transcribed into cDNA. PCR was performed at the following reaction conditions: 1× reaction buffer (200 mmol/l Tris · HCl, pH 8.4, 500 mmol/l KCl), 0.4 mmol/l each dNTP, 1.5 mmol/l MgCl2, 2.5 U Taqpolymerase (GIBCO BRL), 20 pmol of each primer, and 1.5 μl template in a total volume of 25 μl. Using a Mastercycler gradient (Eppendorf, Germany), 34 cycles were run with denaturing for 30 s (after an initial denaturing for 2 min at 94°C) at 94°C, annealing for 1 min at 60°C, and elongation for 2 min at 72°C. Finally, there was an elongation time of 15 min at 72°C. Specific primers were derived from bovine kidney cell line NaPi IIb mRNA sequence (GenBank accession no. X81699): sense 5′-atggtggcctcctcactgctg-3′ (nucleotides 609–629) and antisense 5′-tggggtcatagcagacgtgaa-3′ (nucleotides 1406–1429). A band of 819 bp could be detected by agarose gel electrophoresis, which was identified by cloning and sequencing (sequenced by Agowa, Berlin, Germany).
Northern Blot Analyses
Semiquantitative detection of specific NaPi II mRNAs of jejunum, kidney, and parotid gland was performed as described in detail elsewhere (9). In short, total RNA from kidney cortex and poly(A)+ RNA from parotid gland and jejunal mucosa were fractionated in 1.0% formamide/agarose gels and transferred by capillary blotting onto nitrocellulose membranes. Fixed mRNAs were hybridized to radioactively labeled NaPi II- and β-actin-specific probes. The membranes were analyzed after exposure to a phosphorus imager screen for 2–4 h with a phosphorus imager system (Bio-Rad). The relative amounts of specific mRNA were quantified by reference to β-actin as an internal standard using the quantification software Quantity One (Bio-Rad).
Western Blot Analyses
Semiquantitative detection of NaPi IIb protein in brush-border membranes (BBMs) of jejunal enterocytes was performed as described elsewhere (9). The anti-NaPi IIb antibody was kindly provided by Prof. Dr. J. Biber and Prof. Dr. H. Murer, Institute of Physiology, University of Zürich-Irchel, Zürich, Switzerland. Bands were analyzed quantitatively using the Quantity One software.
Preparation of BBM Vesicles and Uptake Measurements
BBM vesicles (BBMV) were prepared from renal and jejunal epithelia with a modified Mg2+-EGTA precipitation method, and Pi uptake into BBMV was quantified using the rapid filtration technique as described for jejunum and kidney (14,15). Enrichment of the BBM preparations was determined by marker enzyme activities [alkaline phosphatase (AP) for BBM; Na+-K+-ATPase for basolateral membranes]. The used standardized preparation method resulted in nearly pure BBM without endoplasmic contaminations (14). Protein concentration was assayed utilizing the DC method from Bio-Rad (Hercules, CA). Vesicular Pi transport was analyzed in the presence of an inwardly directed Na+ gradient [extravesicular buffer (in mmol/l): 100 NaCl, 100 mannitol, 10 HEPES-Tris, pH 7.4; intravesicular buffer (in mmol/l): 100 KCl, 100 mannitol, 10 HEPES-Tris, pH 7.4]. To calculate the Pidiffusion across the BBM, Pi uptake was measured without an inwardly directed Na+ gradient at an extravesicular Pi concentration of 0.1 mmol/l. The diffusional part of Pi uptake was subtracted from the total Piuptake, resulting in net Na+-dependent Pitransport. Kinetic parameters V max(nmol · mg protein−1 · 10 s−1) andK m (mmol/l) for Na+-dependent Pi transport in kidney and jejunum were calculated from the Michaelis-Menten kinetic for Pi uptake into the BBMV (14).
Determination of Plasma and Salivary Pi
Pi concentration in plasma and saliva was determined colorimetrically using the vanadate-molybdate method (10).
Values were given as means ± SD or SE, withn = number of animals (Tables 1 and2). Significance of differences between plasma and saliva Pi concentrations and kinetic parameters of Na-Pi transport was tested by one-way ANOVA and as a posttest by Tukey's t-test (software Graphpad prism 3.0, San Diego, CA; www.graphpad.com). P values of <0.05 were set to be significant. Results shown in Figs.1-3are given as individual data to illustrate the developmental processes in more detail. Means of blot data (Figs. 1-3, absolute values) are given in Table 2; results of statistical analysis are given in Table 3. All immunodetection and hybridization experiments were performed at least in duplicate. Data of all performed mRNA and protein blots were related to the respective 1- to 7-day values as a 100% basis (Table 2, relative values). All NaPi transport data were correlated with age by linear regression analysis (software Graphpad prism 3.0) (Table 3). For approximation of data in nonruminating and ruminating goats, the absolute and relative values were used, respectively.
Developmental Stages of Animals
The goat kids were allowed to suckle their mothers for the first week of life (group: 1–7 days). Therefore, they were nourished exclusively with colostrum and milk from the first week of lactation. Four- to five-week-old goats (group: 4–5 wk) were also nourished with milk from later lactation by suckling their mothers. The kids were kept with their mothers on straw bedding and so were able to begin eating small amounts of their mothers' straw, hay, or concentrate feed. Eight- to eleven-week-old goats (group: 8–11 wk) were fed ad libitum with milk collected from the mother herd. Because the kids were kept on straw bedding, they ingested straw in amounts that could not be measured quantitatively. Sporadic rumination and measurable salivary secretion were observed in these animals, indicating the onset of weaning. Furthermore, at slaughter the rumen was filled with straw particles. Four- to five-month-old goats (group: 4–5 mo) kept on a concentrate/straw diet expressed physiological rumen activity and an adequate salivary secretion.
Plasma and Saliva Pi Concentrations During Ontogenesis
During the first 4 wk of life, plasma Piconcentrations increased significantly from 2.2 to 3.2 mmol/l and remained constant at this level until the 8th to 11th week. At the fifth month, plasma Pi concentrations markedly decreased to 1.5 mmol/l in ruminating growing goats (Table 1). Neither saliva sampling nor measurement of salivary Pi concentration was possible until the third week of life. At this age, salivary Pi concentration was as high as the plasma Piconcentration and increased continuously up to the 8th to 11th week of life, peaking at ∼40 mmol/l. No further increase occurred (see Fig.3 C) in the 5-mo-old goats.
Jejunal Na+-Dependent PiTransport During Ontogenesis
NaPi IIb mRNA expression in the jejunum of nonruminating goats (1–7 days to 8–11 wk) increased during aging up to the 8th to 11th week of life (Fig. 1, A and B; Tables 2 and3). Simultaneously, NaPi IIb protein expression as well asV max increased during this developmental stage (Fig. 1, C–E; Tables 2 and 3). In ruminating goats (8–11 wk to 4–5 mo of age), mRNA levels decreased with aging. Although the protein expression level remained as high as in that of 8- to 11-wk-old animals, V max decreased during aging. Interestingly, transport capacity (V max) of Na+-dependent Pi transport during the first days of life was as high as in 4–5 mo of age. There was a distinct variation of K m values depending on age.K m values decreased from the 1st until the 11th week of life (Fig. 1 F; Tables 2 and 3), while in ruminating goats there was a significant increase of K m.
Renal Na+-Dependent PiTransport During Ontogenesis
NaPi IIa mRNA expression level did not change in the nonruminating goats (Fig. 2, A and B; Tables 2 and 3). NaPi IIa protein expression could not be investigated; no specific antibody against goat renal NaPi IIa is as yet available. The transport capacity of Na+-dependent Pi transport slightly increased (Fig. 2 C; Tables 2 and 3), but the affinity of the transport slightly decreased (K m values increased; Fig. 2 D; Tables 2 and 3). In ruminating goats NaPi IIa mRNA levels decreased, and, simultaneously,V max also decreased. K mvalues were not affected during this developmental stage.
Pi Transport in the Parotid Gland During Ontogenesis
Using RT-PCR, a NaPi II-specific fragment was cloned from goat parotid gland (Fig. 3 D). The deduced amino acid sequence showed ∼98% homology to the bovine NaPi IIb sequence (accession no.X81699) and ∼66% homology to renal NaPi IIa of sheep (accession no.AJ001385) (Table 4). The subtype of this NaPi II has not yet been determined. Using this PCR fragment as a probe in Northern analysis of goat parotid gland mRNA, a specific band could be detected at ∼1.9 kb (data not shown). Therefore, this NaPi II-specific fragment was used to study its expression in the parotid glands of growing goats. Expression of NaPi II mRNA started at the fourth week of life (the NaPi IIb mRNA/β-actin ratio at 4–5 wk: 0.12) and increased continuously up to the fifth month (the NaPi IIb mRNA/β-actin ratio at 8–11 wk: 0.69 ± 0.09; at 4–5 mo: 2.31 ± 0.73) (Fig. 3, A and B; Table3). Between the 3rd and 11th week of life, the increase of salivary Pi concentrations was accompanied by an increase of NaPi II mRNA expression. Older ruminating goats still expressed higher mRNA levels without a further increase in salivary Piconcentration (Fig. 3 C). In contrast, with PCR, specific bands could also be detected in the 1-wk-old parotid glands (Fig.3 D), indicating that Northern sensitivity was insufficient for detection of NaPi II mRNA at this developmental stage.
The high Pi demand of newborn precocial species such as goats must be balanced by adequate intestinal Piabsorption and renal Pi reabsorption to maintain a positive P balance during ontogenesis. Na+-dependent Pitransport in jejunum, kidney, and parotid gland was studied on functional as well as on molecular levels from the first day until 5 mo of age. The ontogenic changes documented here are among the so-called “specific changes,” i.e., changes in the turnover rate, the site density of transporters, the affinity constant, and/or the expression of different isoforms of transporters (13). Nonspecific ontogenic changes, such as changes in epithelial surface area, proliferation and migration of epithelial cells, phospholipid composition and fluidity of plasma membrane, and paracellular permeability, are also very relevant mechanisms of ontogenetic development (13). Additionally, nephron loss in the kidneys from the juxtamedullary region, changes in proximal tubular cells, such as decreased numbers of mitochondria and reduced basolateral Na+-K+-ATPase activity, could also contribute to the decrease of renal Pi reabsorption capacity during aging (12). These mechanisms were not determined in the present study but have to be kept in mind as additional factors involved in adaptational processes. Ontogenic changes are normally irreversible as opposed to adaptational changes in response to external factors such as P supply. Mature polarized epithelial cells are an essential prerequisite for transport processes in the context of ontogenic changes. Therefore, expression of transporters is strongly correlated to brush border formation (5,18).
Establishment of the Endogenous Pi Cycle During Ontogenesis
The increase of salivary Pi level and NaPi II mRNA expression starting at the third week of life indicates that endogenous Pi recycling has begun even though the rumen is still poorly developed due to the milk diet and before stimulation of salivary secretion rate by solid feed intake. This indicates the presence of at least in part genetically determined NaPi II expression in the parotid gland. At the 8th to 11th week of life, salivary Pi concentrations had reached the levels of adult goats. Although the intake of solid feed was not measured in this study, it must be assumed that due to its increasing intake at this age, salivary secretion was strongly stimulated mechanically. The ability to concentrate Pi in the saliva, possibly by a Na+-Pi type II cotransporter, in connection with the high salivary secretion rate demonstrated the onset of the endogenous Pi cycle at this developmental stage. Surprisingly, the enhanced outflow of Pi from the plasma Pi pool into the growing ruminal compartment did not affect plasma Pi concentrations, which were identical to those of 4- to 5-wk-old milk-fed goats. Therefore, intestinal Piabsorption and renal Pi reabsorption must have been adjusted to meet the Pi needs for endogenous recycling. The increase in V max of Na+-Pi transport during the nonruminating period up to weaning in the jejunum as well as in the kidney strengthened this assumption. Thus the nonruminant period seemed to be an important phase for maturation of intestinal Pitransport. This is in close agreement with the progressive intestinal alteration of mucosal morphology and cell migration velocity observed in suckling lambs from the first to the eighth week of life. While migration rate of cells was highest in newborn lambs, resulting in a short epithelium renewal time of 2–4 days, the migration rate decreased during nonruminating period and renewal time was prolonged (2). This longer life span of enterocytes could contribute to the increasing Na+-Pi transport expression during suckling period. Additionally, the bioavailability of Pi in the solid feed could be reduced compared with that in milk (1). Thus the increase in the intestinal Pi transport capacity could also be due to a stimulation of Pi transporter expression in response to restriction of available dietary Pi in terms of an adaptational process as in adult goats (9).
In newborn goats, K m values of intestinal Pi transport were significantly higher than in all other age groups, indicating a lower affinity for Pi at this developmental stage, although transport capacity was as high as in ruminating goats. Because in adult goats NaPi IIb protein expression is correlated with transport capacity (9), newborn goats should express a lower transport capacity due to low NaPi IIb protein content in the BBM. Existing high transport capacity implies that in the first week another type of Na+-dependent Pitransporter could be present in the jejunum to protect the newborn kid from insufficient intestinal Pi absorption until ontogenetic maturation of the NaPi IIb-mediated Na+-Pi transport. From the 4th until the 11th week of life, jejunal Na+-Pi transport is mediated by NaPi IIb in young goats.
The magnitude of renal Pi transport capacity was much higher in goats than in monogastric species such as rats at corresponding developmental stages (17), indicating that a higher renal Pi reabsorption capacity was expressed even in newborn goats and persisted during aging. Fetal sheep kidney development was finished at 18–90% of gestation (21), resulting in mature glomeruli at day 140of gestation (8). If this is also true for goat kidney development, the prerequisite was given for the expression of Na+-coupled Pi transport across the apical membranes of tubular cells in newborns. Because highV max values result in a renal Pireabsorption capacity of ∼99% in adult goats (20), the comparable high Pi transport capacities in newborn and young goats imply the early availability of this high Pireabsorption capacity. Increasing Pi transport capacity in nonruminating goats could also partly depend on the increasing glomerular filtration rate (GFR) observed in developing sheep (11). Higher GFR resulted in an increase of glomerular filtrated Pi, and to maintain the high tubular reabsorption capacity, Pi transport expression has to be enhanced.
Four- to five-month-old goats fully adapted to solid feed were characterized by significantly reduced plasma Piconcentrations due to their decreasing metabolic demands (16). Pi transport capacities in jejunum and kidney were significantly reduced compared with nonruminating goats. This has also been described in monogastric animals at comparable developmental stages (4, 17). It is unclear whether the reduced intestinal Pi absorption or the diminished renal Pi reabsorption is a primary reason for establishing lower plasma Pi levels. Likewise, it is unclear which mechanisms regulate these changes. Hormones such as thyroid hormone and growth hormone could have a strongly stimulating effect on Na+-Pi transport processes and are closely involved in the postnatal development of mammalians (1). Other hormones and changes in dietary Pi supply and in the extent of cellular Pi-consuming processes may also play important roles in the complex regulation of P homeostasis during ontogenesis.
In these ruminating goats, NaPi IIb expression was studied during ontogenesis because this transporter is mainly responsible for jejunal Na+-Pi transport in adult goats (9). In 4- to 5-mo-old goats, NaPi IIb protein level was as high as in 8- to 11-wk-old goats, but the specific mRNA level was significantly reduced. This could be for several reasons. First, because the life span of enterocytes is reduced in older organisms (13), higher mRNA turnover rates could cause lower transcript levels. Second, the prolonged life span of the transporter proteins in the apical membrane could also contribute to high NaPi IIb amounts at low mRNA levels in adult goats. Third, nutrient transport is restricted to the upper villus region in the older intestine, so that the number of mature enterocytes is decreased, resulting in lower mRNA levels (6, 13). Then an unchanged protein level would indicate a posttranscriptional mechanism to adjust the Pitransport to the Pi demand at this age. Decrease of the NaPi IIb affinity may explain the decrease in transport capacity despite unchanged NaPi IIb protein contents. Decreased NaPi IIa expression in the kidney could correspond to the ontogenetically reduced plasma Pi levels. Because this resulted in lower Pi concentrations in the renal ultrafiltrate, smaller amounts of specific transporters are necessary to maintain ruminants' high renal reabsorption capacity of ∼99% (20).
NaPi II mRNA expression in the parotid glands detectable by Northern blot increased from the fourth week of life. However, both function and cellular localization of the parotid NaPi II transporter are still unknown. The developmental change from weaning to fully ruminating goats was characterized by a strong increase in NaPi II mRNA levels in the parotid gland at unchanged salivary Piconcentrations. The amount of salivary Pi secreted into the rumen is correlated to the salivary flow rate. Therefore, the 4- to 5-mo-old goats should have a higher Pi secretion rate due to greater mechanical stimulation in response to intake of solid feed. Furthermore, to maintain the high salivary Piconcentrations of about 30–40 mmol/l, there had to be an increased amount of Pi transporters (NaPi II) expressed in this age group. This also confirms the role of parotid NaPi II for Pi concentrating in saliva.
In conclusion, P homeostasis of growing goats was maintained from the first day of life by an adequate expression of epithelial Na+-Pi transport capacities in jejunum and kidneys. The special P needs of ruminants due to their rumen ecosystem and their endogenous Pi cycle were satisfied by high intestinal Pi absorption continuously expressed during aging. The high renal Pi reabsorption balanced the high Pi outflow into the rumen when salivary secretion began at weaning. However, newborn and suckling goats were also capable of withholding high amounts of Pi due to the pronounced renal reabsorption capacity. This ability supports the high bone mineralization rate essential in precocial species during ontogenesis.
We thank Prof. W. v. Engelhardt and J. McAlister for critical comments on and correction of the manuscript.
We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft.
Address for reprint requests and other correspondence: K. Huber, Dept. of Physiology, School of Veterinary Medicine Hannover, Bischofsholer Damm 15/102, D-30173 Hannover, Germany (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 10, 2002;10.1152/ajpregu.00357.2002
- Copyright © 2003 the American Physiological Society