Avian kidneys have loopless and looped nephrons; a countercurrent multiplier mechanism operates in the latter by NaCl recycling. We identified an aquaporin-2 (AQP2) homolog in apical/subapical regions of cortical and medullary collecting duct (CD) cells in kidneys of Japanese quail (q), Coturnix japonica. We investigated whether undernutrition during the embryonic/maturation period retards kidney and AQP2 development in quail and programs impaired volume regulation in adults. Protocols included 1) time course and 2) effects of 5–10% egg white withdrawal (EwW) or 48-h post-hatch food deprivation (FD) on nephron growth and qAQP2 mRNA expression, and 3) effects of EwW and FD on qAQP2 mRNA responses to 72-h water deprivation in adults. In metanephric kidneys, qAQP2 mRNA is expressed in medullary CDs at embryonic day 10; distribution and intensity increase during maturation. The number and size of glomeruli continue to increase after birth, whereas nephrogenic zones decrease. In EwW embryos, qAQP2 mRNA expression is initially delayed, then restored; birth weight and hatching rate are lower than in controls. Adults from EwW embryos and FD chicks have fewer (P < 0.01) glomeruli. Water deprivation reduces body weight more in EwW birds than in controls. The results suggest that qAQP2 evolved in metanephric kidneys and that undernutrition may retard nephrogenesis, leading to impaired adult water homeostasis.
- fetal programming
- Japanese quail
- urine concentration
- prenatal nutrition
- birth weight
birds are the only vertebrates other than mammals that can concentrate urine in adaptation to terrestrial environments (7, 8). Avian kidneys contain both loopless reptilian-type nephrons and mammalian-type nephrons that have a loop of Henle running parallel to the collecting duct (CD), enabling it to produce hyperosmolar urine by a countercurrent multiplier system. We have demonstrated that avian kidneys concentrate urine by recycling a single solute (NaCl) (22, 25); their urine-concentrating ability is, however, generally lower than that of mammals (7). Furthermore, the effect of vasotocin [AVT; avian antidiuretic hormone (ADH)] on the water permeability of medullary CDs is only modest (26), and there is no contribution of urea to urine concentration.
Aquaporins (AQPs) are a family of small, hydrophobic proteins originally cloned (for review, see Refs. 1, 39) in mammalian lens. In mammalian tissues, AQP2 is expressed on the apical membrane and subapical endosomes of the principal cells in the CDs (12) and plays an essential role in water conservation. We have identified and characterized homologs of AQPs 1, 2, and 4 from quail (q) kidneys (24, 41, 42). The expression of qAQP2, localized in apical and subapical regions of CD epithelial cells, is modestly enhanced by water deprivation or AVT, more clearly in looped mammalian-type nephrons (18).
Increasing evidence suggests that the onset of diseases in adulthood may originate in adverse events in fetal life (11, 14). Reduced nutritional supply and hypoxia in the perinatal period result in smaller birth size and may predispose humans and experimental animals to various health problems after maturation, including hypertension, type 2 diabetes, and cardiovascular disorders (13–15). The kidney is a primary organ for regulating blood pressure and blood volume homeostasis and may have an essential role in fetal programming of adult diseases (23, 38). It has not been studied, however, whether inadequate supply of nutrients and the resultant retarded renal growth during development lead to impaired volume homeostasis in adult life. Birds provide a unique model for examining the effect of reduced prenatal nutrition on adult function, because their embryos are free of maternal nutritional and hormonal influence.
We therefore investigated the hypothesis that inadequate prenatal nutrition and its accompanying stress impair the structural and functional growth of the kidney, leading to impairment of volume regulation in the adult. Specifically, we examined 1) the time course of nephron development and appearance of qAQP2 mRNA expression during embryonic and neonatal stages, 2) the effect of an inadequate nutrient supply during development on nephron growth and qAQP2 mRNA expression, and 3) whether undernutrition during development/maturation impairs volume regulation in adult life.
MATERIALS AND METHODS
Animals and Maintenance
Fertilized eggs of Coturnix japonica were purchased from a commercial hatchery (Toyohashi, Aichi, Japan) and were incubated in a temperature (37.5°C)- and humidity (60%; 80% 3 days before hatch)-controlled incubator (Ehret egg incubator; AFOS, Haslingden Rossendale, UK) (22, 25). Hatched birds were kept in a temperature (25°C)- and humidity-controlled brooder (JQ-TECH; Toyohashi) for 3 wk and then in a group pen. Chick food (Nisshin-Marubeni, Aichi, Japan) containing 25% protein (minimum) and 0.9% calcium was fed to the birds for 2 wk and gradually replaced with adult food containing 24% protein and 2.5% calcium. Drinking water containing multiple vitamins (GQF Manufacturing) was given for the first 7 days, followed by tap water ad libitum. The photoperiod (12:12-h light-dark cycle) was controlled.
Anesthesia and Arterial Cannulation
Adult quail were anesthetized with pentobarbital sodium (35 mg/kg im for treated group, 50 mg/kg im for intact control group; Abbott Laboratories, North Chicago, IL) supplemented by xylocaine (Astra USA, Westborough, MA). The left ischiadic artery was cannulated with tapered polyethylene tubing, and blood (∼1 ml) was collected into a polyethylene tube containing 10 μl of heparin solution (50 U/ml in PBS, pH 7.4). While the bifurcation of the abdominal aorta and the right ischiadic artery were manually occluded, 5 ml of PBS were quickly infused, followed by a fixative [4% paraformaldehyde (PFA)/PBS, pH 7.2; 20 ml]. Rapid bleaching of the left kidney indicated successful perfusion. Kidneys were quickly removed and immersed in 4% PFA/PBS for 3 days.
Before the experiments, intact eggs were boiled to estimate the approximate volume of egg white. Whites and yolks were separated, and the average percentage of egg white over whole egg weight was calculated (57.8 ± 0.6%, n = 7).
Two treatment methods were used. First, 5–10% egg white (calculated from egg weight by using the above-mentioned average fraction of egg white) was withdrawn before initiation of hatching (referred to as egg white withdrawal, EwW). A sterile G19 blunted needle was inserted through a small hole in the eggshell, and the egg white was withdrawn into a 1-ml syringe. Sham control eggs received a hole puncture and needle insertion without the removal of egg white. Second, birds were deprived of food at neonatal day 4 (D4) for 48 h. Water was given ad libitum.
Body Weight, Plasma Osmolality, Plasma Electrolytes
The body weight (BW) of quail chicks was periodically measured to assess the growth rate. Plasma osmolality was measured by freezing-point depression, using the type OM-6040 osmometer manufactured by Dai-ichi Kagaku (Kyoto, Japan). All the electrolytes were determined using ionic electrodes and an automated electrolyte analyzer, type EA06R (ATWill, Yokohama, Japan).
Following quail decapitation, kidneys from embryos and chicks were quickly excised and immersed in 4% PFA (pH 7.2) for 3 days, dehydrated in ethanol, and embedded in paraffin. To maintain their gross structure, excised kidneys were placed on filter paper before fixation. Whole embryos were collected at embryonic day 5 (E5), whereas kidneys and the soft bones underneath were removed from E10 embryos. Left and right kidneys were collected from E15 embryos and postnatal birds. Tissues were sliced (longitudinal sections) to a thickness of 3–4 μm and stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) for morphological examination. Tissue slices (longitudinal sections, 8 μm thick) were used for in situ hybridization (ISH). Histological examination was conducted with an Olympus model BX50 microscope (Olympus America, New Hyde Park, NY).
In Situ Hybridization
Two cDNA fragment riboprobes (515 and 500 bp) were prepared for constructing digoxigenin-labeled riboprobes. Briefly, the expected fragments of qAQP2 cDNA (1–515 for probe 1 and 388–887 for probe 2) (Ref. 42; GenBank accession no. AY430098) were amplified by polymerase chain reaction (PCR) using two sets of forward and reverse primers (probe 1: forward, 5′-GACTCCTTGCAGCCTCCATG-3′; reverse, 5′-AATGGACAGAGCAGGAGAC-3′; probe 2: forward, 5′-ACAATGAGACGACGACAGG-3′; reverse, GGCCGCTCTCTCAGCTCCTCTCC-3′). The PCR products were cloned into a PCR-4-TOPO cloning vector (Invitrogen Life Technologies; Carlsbad, CA), screened, and purified with Mini-prep (Qiagen, Valencia, CA) for sequencing. We selected plasmids that contain a cDNA sequence in which antisense and sense (negative control) probes can be prepared from the T7 RNA polymerase site. The selected plasmids were further processed using Medi-prep to obtain a sufficient amount of cDNA. The plasmids with qAQP cDNA fragments were linearized with SpeI for both sense and antisense probes. The template (500 ng) was then transcribed in vitro by incubation with T7 DNA-dependent RNA polymerase for 2 h and was labeled with a digoxigenin (Dig) RNA labeling kit (Roche Diagnostics, Penzberg, Germany) following the manufacturer's protocol.
ISH was done using the automated mRNA ISH application (Ventana Medical Systems, Tucson, AZ) (2, 27). Briefly, serial sections were automatically deparaffinized, fixed, and acid treated. The tissue sections were then subjected to cell conditioning and protease digestion. Hybridization was performed with a Dig-labeled qAQP2 antisense probe (1–5 ng/slide) for 17 h, followed by incubation with an alkaline phosphatase-conjugated anti-Dig antibody (Roche Diagnostics) at 37°C for 1 h; the signal was detected using a nitro blue tetrazolium chloride 5-bromo-4-chloro-3-indolyl phosphate toluidine salt substrate solution for 6 h. qAQP2 mRNA signals were equally well detected by the two hybridization probes derived from the differing locations of qAQP2 cDNA mentioned above with no nonspecific signals. The probe (designated qAQP2 mRNA probe 1) was used for further study, and ISH was repeated twice on each tissue slice using the independently labeled probes. Following preliminary studies examining the dose response of signal intensity, we selected 5 ng/slide for the time-course study to ensure visibility of signals during development. A lower dose, 1 ng/slide, was selected for comparing the level of expression among the four treatment groups, since a lower dose might reveal possible differences, if any, more clearly.
Protocol I: Time course of kidney development and AQP2 mRNA expression in intact eggs.
Kidneys (whole embryos at E5, n = 7) were collected at different developmental stages, including E5 (mesonephros), E10 (n = 11), and E15 (n = 8) (mesonephros and metanephros), postnatal day 2 (D2; neonate, n = 8) and D10 (n = 7), and 3 wk (maturing, n = 9) and 5 wk (adult, n = 6). Body growth, structural development of kidneys (HE and PAS staining), and qAQP2 mRNA expressions and distributions (ISH) were examined.
Protocol II: Time course of kidney development and AQP2 expression in eggs with undernutrition.
Fertilized eggs were assigned to two groups: 1) sham treatment (SH) and 2) 5–10% EwW. Kidneys were collected from E5 (whole embryos, n = 5–6), E10 (n = 2–6), and E15 (n = 6–7) embryos and from newborn chicks (D2, n = 5). Body growth, structural development of kidneys (HE and PAS staining), and qAQP2 mRNA expressions and distributions (ISH) were examined.
Protocol III: Effect of undernutrition during development on water balance and AQP expression in adulthood.
Fertilized eggs were assigned to three groups: 1) intact control (CT), 2) sham treatment (SH), and 3) 5–10% EwW. At D4 after hatching, one-half of the CT group underwent food deprivation for 48 h (FD). The EwW and FD groups are referred to as having “undernutrition” treatment. Birds were kept in groups (CT, n = 10; SH, n = 17; EwW, n = 4; FD, n = 8), and BW was determined periodically in all groups. The EwW group had a small number due to low hatching rate. At 4.5 wk, each group was divided into two subgroups and processed for 1) normal hydration and 2) water deprivation for 72 h. Blood was collected either by decapitation or via a catheter inserted into the left ischiadic artery under anesthesia. Right and left kidneys were quickly removed in one piece, placed on filter paper, and immersed in 4% PFA (pH 7.2). In catheterized birds, kidneys were fixed by in vivo perfusion of the fixative.
Assessment for kidney development was conducted in longitudinally cut slices by 1) counting the number of glomeruli in a designated cortical (superficial) area (0.2 mm2, 5 areas per kidney slice) and in a deeper zone (1 mm2, 2 areas per kidney slice) and 2) determining the size of the glomerulus (4–5 glomeruli per kidney slice representing various parts of the kidney; glomeruli containing a vascular pole or a Bowman's capsule transit to a proximal tubule were selected). Glomeruli were photographed (Olympus digital camera model C-2020) and then measured with the NIH Image program. The size of the “differentiating nephrogenic” region on the cortical surface and its proportion to the entire medullary cone were also examined. We measured the entire medullary cone rather than the cortical area, because in avian kidneys the zonation between cortex and medulla is not as clear as in mammalian kidneys. At least three to four regions were examined per kidney. Averaged values were used to represent a bird for a respective index.
For evaluation of hybridized signals of qAQP2 mRNA, two factors were considered for evaluation: 1) the number of CDs in the designated area in which qAQP2 mRNA was positively expressed and 2) the degree of intensity of the signals. We set scores (from 1 to 6) according to the distribution and intensity of the mRNA signals in epithelial cells in CDs in superficial and deeper areas: 1 = no labeling, 2 = faint labeling in a few cells per CD or in a few CDs per medullary cone, 3 = medium intensity labeling and distribution, 4 = medium-strong intensity labeling and distribution, 5 = strong intensity labeling in majority of CDs; and 6 = very strong signal in all CD cells. Assessment was done by two individuals in a blinded test fashion. Initially, we evaluated ISH by counting the number of positively labeled CDs in 1) the deeper medulla (stem part of CD), 2) the medullary CD branch area (number of CDs per mm2), and 3) the superficial (cortical) regions (number within unit area). At least five areas of each region were examined. The results of this morphometric method, however, did not agree with the findings from visual observation and hence were not used.
All data are means ± SE. For statistical analysis, a one- or two-factor ANOVA was used, followed by Tukey's honestly significant difference unbalanced test and post hoc comparison when applicable. The difference between control and experimental groups was determined using Student's t-test in some analyses (see legends). The difference was considered significant at a P value <0.05.
Time-Course Development of Nephrons (Protocol I)
At E5, mesonephric kidneys with larger glomeruli (area: 6,273 ± 728 μm2, n = 6) were seen; at E10, both meso- and metanephric kidneys were present (Fig. 1A). In E15 embryos, mesonephric kidneys were scarce, and after hatch, only metanephric kidneys were seen. In metanephros, glomeruli were larger in deeper regions than in superficial regions (Fig. 1B). The number of glomeruli per kidney increased exponentially during the later embryonic stage and early postnatal days (Fig. 2A, left) and maintained a similar level after D10 (Fig. 2A, right). In maturing kidneys, the number of glomeruli in the superficial (cortical) area was higher than in the deeper region (Fig. 2A, right). In contrast, glomeruli were smaller in the superficial regions. Whereas the glomeruli were of similar size in the E15 to 5-wk age groups in the superficial regions, glomeruli in the deeper region were larger and showed an age-dependent increase (Fig. 2B). Elongation of medullary cones continued ∼4 wk.
Throughout the surface area of developing metanephric kidneys (E10, E15), condensed areas could be seen that contained PAS-positive dark-stained mesenchymal cells (Fig. 3A). In this mesenchymal area, no proximal tubules identified by brush borders were seen. In contrast, in the inward, more mature regions, proximal tubules consisting of columnar epithelial cells with brush border and distal tubules that showed cuboid-shaped cells with open lumina were seen. The size of the condensate zone (percentage of medullary cone area, longitudinal section) decreased with maturation. Condensate was still detectable at 3 wk but not at 5 wk after birth (Fig. 3B).
Time-Course Appearance of qAQP2 mRNA (Protocol I)
Clear and specific qAQP2 mRNA signals were detected in cortical and medullary CDs by ISH of Dig-labeled riboprobes. Riboprobes derived from two fragments (∼500 bp) of the qAQP2 cDNA showed similar hybridization results. No positive mRNA signals were detectable by sense probes in any age group. No apparent differences were observed in the appearance of qAQP2 mRNA signals between perfused and nonperfused (directly immersed in fixative) kidneys.
No qAQP2 mRNA expression was detected in mesonephric kidneys (examined at E5, E10, and a part of E15) by ISH of a DIG-labeled riboprobe. In E10 metanephric kidneys, qAQP2 mRNA signals were visible in some epithelial cells, presumably in CDs from deeper medullary regions (Fig. 4A). In kidneys from E15 chicks, ISH signals were more clearly seen and were localized in large medullary CDs and their major branches (Fig. 4B). In newborn and maturing chick kidneys, the intensity and distribution of expression increased (Fig. 5, A and B). In kidneys from 3-wk-old maturing quail, strong hybridization signals were seen along the CDs and their branches in the medulla and superficial (cortical) regions (Fig. 5C). It therefore appears that AQP2 evolves while active nephrogenesis is taking place and that its expression continues to increase while the renal architectural structure and renal tubules are maturing after birth. In cortical CDs from mature birds, a rather abrupt transition of regions from qAQP2 mRNA positive to qAQP2 mRNA negative was noted (Fig. 5D).
Effect of Undernutrition Treatments on Hatching Rate, Birth Weight, and Body Growth (Protocols II and III)
Chicks from EwW eggs had a lower birth weight (6.52 ± 0.19 g; P < 0.05) than intact controls (CT; 7.14 ± 0.21 g) (Fig. 6A). The hatching rate of SH (35.0%, n = 20) and 5% EwW eggs (23.1%, n = 26) was lower than that of CT eggs (68.8%, n = 16). BW, however, similarly increased as a function of time in both intact and treated eggs (Fig. 6B). Autopsy revealed that many embryos in unhatched eggs died in middle to late, rather than early, stages of embryonic life. The bird group (FD) that was deprived of food for 48 h during the neonatal period (D4–D6) lost BW significantly (from 16.6 ± 0.5 to 11.4 ± 0.4 g) during the food deprivation period but were restored to control levels by 3 wk of age (Fig. 6B).
Effects of Undernutrition Treatment on Development of Kidney and qAQP2 mRNA Expression (Protocol II)
At E10, embryos from the EwW group had wider nephrogenic zones (20.4 ± 1.3% of medullary cone, P < 0.01) near the surface of the kidney than SH did (10.1 ± 2.4% of medullary cone) and more abundant mesonephric areas than were seen in E10 embryos from intact or sham control eggs. At E15 and D2, mesonephros was still clearly seen in the EwW group but not in the CT group. Kidneys from the EwW birds were smaller than those of CT birds at D2 (by observation, not measured). Glomeruli in the SH and EwW birds were similar in number at E15 (SH: 209.8 ± 24.7 glomeruli/mm2, n = 4; EwW: 226.2 ± 33.5 glomeruli/mm2, n = 6) and D2 (SH: 855.2 ± 70.0 glomeruli/mm2, n = 5; EwW: 731.4 ± 70.8 glomeruli/mm2, n = 5); glomerular numbers were comparable to those of the normal CT group (Fig. 2A). Likewise, no apparent difference was noted between SH and EwW groups in the size of glomeruli at E15 (SH: superficial, 577.2 ± 50.5 μm2; deeper, 1,214.0 ± 173.9 μm2, n = 4; EwW: superficial, 572.1 ± 63.5 μm2; deeper, 1,349.3 ± 169.4 μm2, n = 5) and D2 (SH: superficial, 587.4 ± 28.8 μm2; deeper, 1,709.8 ± 122.8 μm2, n = 5; EwW: superficial, 546.9 ± 26.5 μm2; deeper, 1,515.4 ± 129.5 μm2, n = 5). Kidneys were not collected after D2 in this protocol because of the limited number of hatched birds. In EwW kidneys, no qAQP2 mRNA signal was detectable at E10 (Fig. 6C). In kidneys from E15 embryos, signals were primarily localized in the large medullary CDs, similar to the case in CT kidneys. In D2 chick kidneys derived from EwW eggs, qAQP2 mRNA signals were present in both the medullary and cortical regions, as in CT kidneys.
Effects of Undernutrition During Development on Water Balance and qAQP2 mRNA in Adult Quail (Protocol III)
The number of glomeruli in superficial areas of kidneys from normally hydrated quail at 4.5 wk of age was significantly lower in the EwW and FD groups than in CT birds (Fig. 7A), although no apparent difference was detected in the histological examination or size of nephrons (data not shown). The changes in BW (%) of the birds subjected to 72 h of normal hydration or water deprivation are summarized in Fig. 7B. BW loss was significantly greater in birds hatched from EwW eggs. Chicks that were deprived of food for 48 h during the neonatal period showed a similar tendency, but the difference was not significant. Plasma osmolality increased significantly in all groups (Table 1). Plasma Na+ and Cl− levels also showed a tendency to increase after water deprivation, but no change was noted in plasma K+ level (Table 1).
The distribution and intensity of qAQP2 mRNA expression were graded from 0 (no signal) to 6 (highest) according to a set scale (see materials and methods). The results are summarized in Fig. 8 (preliminary study). In normally hydrated groups, qAQP2 mRNA expressions tended to be higher in undernutrition treatment groups than in control groups. In the intact and sham control groups, qAQP2 mRNA signal scores were significantly higher in both the medulla and cortex in water-deprived birds. In birds subjected to undernutrition, no further increase was noted by water deprivation.
During vertebrate evolution, three types of kidneys (pronephros, mesonephros, and metanephros) evolved that can serve as developmental models (16). In developing avian kidneys (for review, see Ref. 10), the pronephros is formed around the second day of incubation but disappears early. The opisthonephros (mesonephros) differentiates from the nephrogenic cord (segment 16–24) and reaches its full extent at E5–E6. We noted in E5 quail kidneys a nephric structure that contained large glomeruli and renal tubules similar to proximal tubules. The distinction between pro- and mesonephric kidneys, however, was not clear. Mesonephric kidneys are functional until hatching but cannot excrete excess salt loading by producing hyperosmolar urine (44). The adult metanephric kidney develops from the blastema of the posterior nephrogenic cord around segment 28 (10). In mammalian kidneys, signals from the metanephric mesenchyme appear to initiate kidney development by inducing formation of the ureteric bud from the Wolffian duct (3, 16). Subsequently, the growing ureteric bud also starts to branch in response to the mesenchymal signals and in turn transmits signals that induce mesenchymal cells to condense and generate pretubular aggregates around the branches of the ureteric tree (32). In quail metanephric kidneys, we noted mesenchymal condensation near the surface. This area spread throughout the whole surface area at E10–E15, remained considerably after birth, and gradually decreased with maturation, suggesting that, as in the mammalian kidney, this condensate area may be important for nephrogenesis. Furthermore, glomeruli increased in size and number during development and after birth for 2–3 wk, indicating that nephrogenesis had not been completed at birth. Also, glomeruli were smaller in the outer zone than in the inner zone of the metanephros and presumably develop into, respectively, loopless (reptilian-type) and looped (mammalian-type) nephrons; this tendency continued in adult kidneys.
AQP2 Expression During Development
In the present study, we could not detect qAQP2 mRNA hybridization signals in mesonephric kidneys (E5, E10, and a part of E15). Positive hybridization signals were seen in the CD/ureteric bud-like structures of metanephric kidney in the deeper area of E10 kidneys and, more clearly, in medullary cones in E15 kidneys. Expression continued to increase during the postnatal period in deeper (medullary) CD branches and then in superficial (cortical) CDs while the medullary cones were elongating. During development, rat kidneys express AQP mRNAs 1, 2, 3, and 4 (Ref. 40; ribonuclease protection assay). The AQP2 mRNA signal is barely detectable at E16 (40), whereas no immunoreactive AQP2 is detected in the branching ureteric buds of E15 fetal rat kidney. At E18, AQP2 and AQP3 are localized in ureteral buds at, respectively, the apical and basolateral sides of epithelial cells (4). The mRNA levels of AQPs 1, 3, and 4 reached maximum at the time of birth, whereas the level of AQP2 mRNA continued to increase postnatally for 10 days (4) and 4 wk (40). This delayed expression of AQP2 in mammalian kidneys agrees with the time-course expression of qAQP2 mRNA we observed in quail kidneys. In adult quail kidneys, we identified qAQP1 (43), qAQP2 (42), and qAQP4 (41). In developing quail kidneys in the present study, the presence of AQPs (either mRNA or protein) other than qAQP2 mRNA was not determined.
The structures and functions of the medulla in avian (24, 26) and newborn rat kidneys are similar (19). Also in both, AQP expressions are lower than in mature animals (Refs. 4, 40; current study). AQP2 expression was increased adequately by dDAVP (less marked than in adults), whereas urine osmolality remained low in newborns (5). After dehydration, AQP2 mRNA and protein levels increased, but the response was lower than in mature rats. Moreover, urine concentration did not increase proportionately, suggesting that the mechanism in signal pathway distal to AQP2 may still be underdeveloped in newborns (5).
Effects of Reduced Nutrition During Development on Nephrogenesis, AQP2 Expression, and Fluid Balance in Adults
The influence of environmental alterations such as temperature change on subsequent development was first reported in avian eggs (Ref. 6; description from Ref. 14). Recent epidemiological evidence and experimental studies in animals indicate that inadequate supply of nutrients, oxygen, and hormones during the fetal period affects organogenesis, programming abnormal cardiovascular and metabolic functions in adults (for review, see Refs. 11, 13, 15). The concept of developmental origins of adult disease has been extensively studied in various species, including humans (14, 34); the timing, duration, severity, and type of insult determine the specific physiological outcome (11). In particular, inadequate maternal diet often causes impaired organogenesis, specifically in the kidney and vascular system, reduces fetal size and birth weight, and in adults causes hypertension and obesity, reduces glucose tolerance (type 2 diabetes), and increases the risk of cardiovascular-renal diseases (14, 15).
Our present study demonstrates that 5–10% EwW before the incubation of Japanese quail eggs reduced hatching rate, birth weight, and kidney size. We noted a larger nephrogenic (condensate) area in E15 EwW embryos compared with control embryos. Also, in contrast to the findings in control embryos, qAQP2 mRNA hybridization signals were not detected in kidneys from EwW embryos at E10, suggesting that nephrogenesis may be somewhat delayed in undernutrition embryos. Furthermore, mature birds from EwW eggs and FD groups exhibited a smaller number of glomeruli. It has been suggested that intrauterine growth retardation caused by maternal low-protein diet results in smaller kidneys and fewer nephrons, leading to 1) high blood pressure, possibly due to suboptimal renal handling of sodium, and 2) compensatory kidney growth and overload of remaining nephrons, such as excess glomerular filtration with resultant glomerulosclerosis (14, 17, 36).
Although the precise mechanism of fetal programming remains unclear, it could occur at the gene, cell, tissue, organ, and whole system levels (14). In this context, the role of an impaired renin-angiotensin system in leading to delayed nephrogenesis (28, 36, 38), the role of excess fetal glucocorticoid occurring in response to a reduction in the energy source (33, 37), and the role of impaired vascular development (20) and excess Na+ transport across renal tubules (21) have been investigated. Experimentally, maternal protein restriction leads to metanephric apoptosis, and the total number of cells in the metanephros is low (35). Induction of molecular changes that may be transgenerational also has been suggested (33). It has been reported that femoral arteries from young chickens exposed to chronic moderate hypoxia during in ovo development showed increased reactivity to electrical stimulation, whereas the effect of nitric oxide synthase blockade by nitro-l-arginine methyl ester was reduced (31). Acetylcholine-induced relaxation decreased in E19 chick embryos exposed to hypoxia, whereas no change was noted for the EwW group, suggesting that endothelial dysfunction may have occurred in the former (30).
In our study, the BW of birds derived from EwW eggs decreased significantly more during 72 h of water deprivation compared with the BW changes in CT, suggesting that the kidney's capability for water conservation was somewhat impaired. The BW reduction of birds that were food deprived 48 h in early postnatal life also showed a similar tendency, although the difference was not significant. No differences were seen in the postnatal growth rate between CT and EwW groups. Although the quail deprived of food for 48 h at an early postnatal day (FD group) showed retarded BW gain initially, it was restored by 3 wk of age. Therefore, the larger weight loss seen in water-deprived reduced-nutrition groups is not due to delayed body growth. Ross and colleagues (9, 29) reported that maternal dehydration in sheep results in low birth weight and that the offspring show high plasma Na+, high osmolality, and hypertension. These offspring showed a less sensitive plasma osmolality threshold with a steeper secretion curve of ADH than normal controls (29). We have no information at present on plasma vasotocin (avian ADH) levels in birds with undernutrition treatment.
Summary and Perspective
First, in quail, nephrogenesis continues after birth; qAQP2 mRNA was detected in metanephric kidneys, and expression progressively increased with nephron development and maturation of the renal architectural structure after birth. Second, quail groups that received reduced nutrition during the nephrogenic period exhibited smaller birth weight, somewhat delayed kidney growth, and, at a young adult age, a decreased number of glomeruli. Third, water deprivation caused greater body weight loss in undernutrition birds than in control birds. Thus, in undernutrition quail, which have a smaller number of nephrons, water balance may be impaired. For control of body fluid homeostasis, the maintenance of glomerular filtration is particularly important for birds, since urine flow is regulated in part by glomerular intermittency (8). In the present study, qAQP2 mRNA levels of normally hydrated undernutrition groups were not lower than those of controls, but no further increase occurred after water deprivation. It is possible that the failure of water deprivation to induce a further increase in qAQP2 mRNA in undernutrition birds led to a greater BW loss. It remains to be determined whether an abnormal level of vasotocin or altered vasotocin receptor responsiveness is responsible for the impaired water balance and the lack of an increase in qAQP2 mRNA after water deprivation. It is also important to determine whether the changes of qAQP2 mRNA noted in the present study correlate with the changes in qAQP2 protein. Fetal programming of adult disease is an important human health issue in which multiple inducing factors and mechanisms are involved. Utilizing avian eggs, we can study the direct effects of environmental factors, such as temperature, oxygen levels, humidity, and nutritional supplies, on embryonic growth. Such studies can provide insight into understanding possible mechanisms of developmental origins of adult diseases.
We are grateful for support by JSPS Fellowship Grant L-005573, National Science Foundation Grant IOB0615359, and National Heart, Lung, and Blood Institute Grant HL-52881 (H. Nishimura), National Kidney Foundation Grant MG61943 (K. Lau), Le Bonheur Small Grant (K. Lau), and JSPS Priority B Grant-in-Aid 17390247 (T. Yamamoto).
We thank Drs. Eishin Yaoita, Utaka Yoshida, and Asako Matsuki, as well as Keiko Yamamoto, for conceptual and technical advice. We also thank Guibin Su and Akira Inoue for excellent technical assistance.
The physiological studies and molecular analyses of the presented studies were conducted at the Department of Structural Pathology, Niigata University School of Medical and Dental Science, Niigata, Japan, during the tenure of the Invitation Fellowship Program of Japanese Society of Promotion of Sciences (JSPS) awarded to H. Nishimura. Preliminary studies were presented at the annual meetings of the Federation of American Societies for Experimental Biology, San Francisco, CA, 2006, and the American Society of Nephrology (Renal Week), San Diego, CA, 2006.
Present addresses: K. Lau, Department of Pediatrics, University of California, Davis, Sacramento, CA; R. Kuykindoll, Division of Natural Science and Mathematics, Department of Biology, LeMoyne-Owen College, Memphis, TN.
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