1Institute of Experimental Clinical Research, Aarhus University, and 3Department of Clinical Physiology, Aarhus University Hospital, DK-8200 Aarhus N; 2The Water and Salt Research Center and 4Institute of Anatomy, Aarhus University, DK-8000 Aarhus C; 5Institute for Basic Psychiatric Research, Department of Biological Psychiatry, Aarhus University Hospital, DK-8240 Risskov, Denmark; and 6Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
Submitted 27 May 2003 ; accepted in final form 9 January 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
rat; obstructive nephropathy; kidney function; magnetic resonance imaging; aquaporin; sodium transporter
In the rat, >90% of nephrogenesis has taken place during the first 10 postnatal days and the final maturation of the renal functions occurs thereafter. Cortical and medullary anatomy matures significantly during the critical period of the 3rd wk after birth (39). Previously, it was demonstrated that urinary tract obstruction during this vulnerable period aggravates renal functional development and is associated with disproportionate renal functional impairment (8), and 3-wk-old rats subjected to unilateral ureteral obstruction (UUO) have a highly activated tubuloglomerular feedback mechanism when studied 36 wk later (33). Moreover, onset of complete UUO at 1419 days of age in rat results in more severe impairment of kidney growth than the impairment in response to neonatal UUO at 15 days of age or during adulthood (11). Furthermore, 3 mo after release of UUO during days 1419, renal growth was decreased by 50%, compared with a 30% reduction after release of UUO during days 15 (11). The number of glomeruli was reduced by 50%, regardless of the timing of UUO, but glomerular size was reduced only in rats with UUO during days 1419 (11). These results demonstrate that, in the period immediately after nephrogenesis, the developing kidney is particularly susceptible to long-term impairment from temporary obstruction, suggesting that a delay in release of severe ureteral obstruction may have a detrimental impact on renal function later in life.
The effect of neonatally induced partial UUO (PUUO) on kidney function during this vulnerable period is not fully understood. Previously, it was demonstrated that glomerular filtration rate (GFR) is reduced in proportion to the severity of the obstruction (21). Recently, using magnetic resonance imaging (MRI), we demonstrated that renal blood flow (RBF) was progressively reduced in rats with long-term follow-up after neonatal PUUO (51). It has been hypothesized that renal functional deterioration in a hydronephrotic obstructed kidney stimulates the growth and function of the contralateral nonobstructed kidney at a faster-than-normal rate and before functional deterioration of the hydronephrotic kidney is detectable (26). Recently, we demonstrated that compensatory growth of the contralateral nonobstructed kidney during a 24-wk observation period is not detectable before the appearance of RBF deterioration in the neonatally obstructed kidney (51), suggesting that compensatory growth is not a useful predictor of early functional deterioration.
Characteristically, a very important sign of urinary tract obstruction is impairment of urinary concentrating capacity and, eventually, development of nephrogenic diabetes insipidus in severe cases (20). Urinary concentration and dilution depend on a discrete segmental distribution of transport properties along the renal tubule: 1) the hypertonic medullary interstitium, which is generated by active NaCl reabsorption in water-impermeable nephron segments and 2) the high water permeability (constitutive or vasopressin regulated) in other renal tubular segments for osmotic equilibration, which chiefly depends on aquaporins (AQPs) (24). Thus defects in any of these mechanisms would be predicted to be associated with urinary concentrating defects.
AQPs are a family of membrane proteins that function as water channels (37). AQP1 is highly abundant in the proximal tubule and descending thin limb (38), whereas AQP2 is the apical water channel of the principal cells and is the chief target for regulation of collecting duct water permeability by vasopressin (35, 36). Water transport across the basolateral plasma membrane of collecting duct principal cells is mediated by AQP3 (15) and AQP4 (47). Consistent with the roles of AQPs in renal regulation of water balance, we recently demonstrated that UUO and bilateral ureteral obstruction cause severe dysregulation of renal AQPs, which is associated with impaired renal water handling (28, 29). UUO was also demonstrated to alter expression levels of renal sodium transporters associated with deranged urinary sodium excretion (30), supporting the view that dysregulation of these transporters plays an essential role in the impaired urinary concentration and sodium handling in response to obstruction.
The aims of this study were therefore to investigate long-term changes in renal functions in response to neonatal PUUO. In particular, changes in RBF, GFR, and renal sodium and water handling of the obstructed and nonobstructed kidney, together with the renal protein expression of major sodium transporter and water channels, were examined using a proteomic approach. Furthermore, we aimed at addressing whether early, rather than late, release can prevent the renal functional reduction in response to neonatal PUUO.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies were performed in female Münich-Wistar rats. Rats were subjected to severe PUUO or sham operation within the first 48 h of life according to a modification of the technique of Ulm and Miller (51). Briefly, newborn rats were anesthetized with ether and placed on a heated table. The left ureter was exposed through a midline incision. A PUUO was created by embedding two-thirds of the left ureter in a psoas muscle tunnel. The sham group was prepared by laparotomy and mobilization of the left ureter. After surgery, rats were kept in an incubator at 30°C until they had totally awakened; then they were returned to the regular animal units with their mothers. At 1 or 4 wk after onset of obstruction, 18 rats from the PUUO group were subjected to a second operation performed under general anesthesia with ether to release the obstruction by removal of the ligatures and suturing of the underlying psoas muscle. At 4 wk of age, the rats were separated from their mothers and housed two per cage. During the experiments, the rats were maintained at controlled temperature (2224°C) and moisture (60%) with a 12:12-h artificial light-dark cycle. The rats were fed a standard rodent diet and tap water. After 24 wk, the rats were killed. The study complied with the Danish regulations for care and use of experimental animals. Rats were allocated to the following groups: 1) the PUUO group (n = 7), in which the animals were subjected to PUUO for 24 wk, 2) the PUUO + 1wR group (n = 10), in which PUUO was released 1 wk after onset of obstruction, 3) the PUUO + 4wR group (n = 8), in which PUUO was released 4 wk after onset of obstruction, and 4) the sham group (n = 11), which consisted of matched sham-operated controls.
MRI examinations were carried out under pentobarbital sodium anesthesia (50 mg/kg body wt ip; Pentothal, Abbott Scandinavia, Solna, Sweden) in all rats at 5, 12, and 24 wk of age. Before the animals were killed, single-kidney function was examined. Then the two kidneys were removed, the wet weight was determined, and the kidneys were frozen for later analysis.
MRI
Briefly, the MRI examinations were performed with a small-bore Sisco 7-T system (Varian, Palo Alto, CA). The rat was placed supine in a Helmholtz 4-cm-diameter head coil and then subjected to an imaging protocol including measurements of single-kidney RBF and total kidney volume (TKV).
RBF Measurements
RBF measurements were obtained using a phase-contrast technique involving a gradient echo sequence with bipolar flow-sensitive gradients. The strengths of the flow encoding gradients were set according to the values from a previous study (44). Ten 1.2-mm-thick slices were prescribed perpendicular to the renal veins. Each slice had a 7 x 7-cm2 field of view and a resolution of 350 x 350 pixels to ensure that blood flow in the renal veins could be derived. Other parameters were as follows: 150-ms repetition time, 5.5-ms echo time, 55° excitation flip angle, and four data averages. Acquired phase images were subtracted, and the vein flow was determined by multiplying by the renal vein area for each available slice. The individual kidney RBF was then calculated as an average of the flow values for all slices.
TKV Measurements
A gradient echo sequence was used to obtain a series of axial slices through the kidney to determine TKV. Depending on the kidney size, 2030 equidistant 1.0-mm-thick slices were employed to sufficiently cover both kidneys. The field of view and pixel size were the same as those described for RBF measurements, and other parameters were as follows: 125-ms repetition time and 4-ms echo time. Postprocessing included manual identification of each kidney for all slices, and by carefully encompassing regions of interest, TKV was measured by the sum-of-areas principle (50).
Measurement of GFR and Tubular Functions
GFR was measured using renal clearance of 51Cr-EDTA at 24 wk after the onset of PUUO. Seven days before the clearance studies, the left femoral artery and vein were catheterized under pentobarbital sodium anesthesia (50 mg/kg body wt ip). The arterial and venous catheters were fixed as described by Petersen et al. (43), sealed with 50% glucose solution containing heparin (500 U/ml) and streptokinase (10,000 U/ml), and fixed. After instrumentation, 5 ml of saline and 10 µl of analgesic (buprenorphine, Temgesic) were given subcutaneously. After recovery from anesthesia, the rats were returned to the animal units and housed individually (49).
Renal clearance of 51Cr-EDTA was measured using a constant-infusion clearance technique. Briefly, the rats were anesthetized as described above and then placed on a heating table to maintain rectal temperature at 37°C. Through a midline incision, both ureters were exposed and catheterized (0.762-mm flexible plastic tubing; Tygon, Weyerhaeuser, Cleveland, OH) for the urine collection. The incision was closed to prevent loss of body fluid. A priming 15-min intravenous dose of 51Cr-EDTA (0.2 MBq) was followed by a sustained infusion (0.005 MBq/min) during a 75-min equilibration period and two 1-h urine collection periods. An intravenous infusion of a 25 mM glucose solution (40 µl/min) was provided simultaneously to maintain an adequate minimum urine flow rate for biochemical analysis of the collected urine. Timed blood samples (150 µl) were taken from the arterial catheter every hour during the urine collection periods and replaced immediately with the same volume of heparinized donor blood. Timed urine samples were gravimetrically collected every hour from both ureters. The plasma and urine samples were diluted, and 51Cr-EDTA was counted using an Auto-Gamma Counting System (COBRA, Packard Instrument, Meriden, CT).
The osmolality of urine and plasma was determined by freezing-point depression (advanced osmometer, model 3900, Advanced Instruments, Norwood, MA, and Osmomat model 030-D, Gonotec, Berlin, Germany). The plasma concentration of sodium was determined (Kodak Ektachem 700XRC). The concentration of urinary sodium was determined by standard flame photometry (model FCM6341, Eppendorf).
The rats were killed after the study. The harvested kidneys were rapidly frozen in liquid nitrogen and kept at 80°C until assayed.
Analysis of Renal AQPs and Sodium Transporters
Membrane fractionation for immunoblotting. Kidneys were minced finely and homogenized in 9 ml of dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, 8.5 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride) with five strokes of a motor-driven Potter-Elvehjem homogenizer at 1,250 rpm. One microliter of this homogenate was used to measure the total protein concentration as previously described (see below) (28). This homogenate was centrifuged in a Beckman L8M centrifuge at 4,000 g for 15 min at 4°C. Gel samples (Laemmli sample buffer containing 2% SDS) were made from this membrane preparation.
Total protein concentration. A bicinchoninic acid (BCA) protein assay kit (Pierce) was used to determine total protein concentration. A fresh set of protein standards (BSA at 2 mg/ml; 0, 1.5, 5, 10, 20, and 30 µl) containing the same component was prepared and used to determine the protein concentration for each sample. Distilled water was then added to all standards and samples to ensure that the total volume was the same in each tube (50 µl). One milliliter of BCA protein assay reagent mix solutions (reagents A and B at a ratio of 50:1) was added, and all tubes were incubated at 37°C for 30 min. All samples were measured at 562 nm using a Helios gamma spectrophotometer (Thermo Spectronic) with the Protein Analysis Pack and the BCA method. A standard curve was made by determination of the protein standards before all the other samples. Each sample was measured in duplicate, and the mean values were used.
Electrophoresis and immunoblotting. Samples of membrane fractions from whole kidney were run on 9 or 12% polyacrylamide minigels (Mini Protean II, Bio-Rad). For each gel, an identical gel was run in parallel and subjected to Coomassie staining (48). The Coomassie-stained gel was used to ascertain identical loading or to allow for potential correction for minor differences in loading after scanning and densitometry of major bands. The other gel was subjected to blotting. After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20, pH 7.5) for 1 h and incubated with primary antibodies (see below) overnight at 4°C. The blots were washed as described above and then incubated with horseradish peroxidase-conjugated secondary antibody (P448, Dako, Glostrup, Denmark; diluted 1:3,000). After the blots were washed for the last time as described above, antibody binding was visualized using the enhanced chemiluminescence (ECL) system (Amersham). ECL films were scanned using a Hewlett-Packard Scanjet scanner and Adobe Photoshop software. The labeling density of blots was determined from samples of kidneys of PUUO, PUUO + 1wR, and sham groups. The labeling density was corrected by densitometry of Coomassie brilliant blue-stained gels (i.e., to control for minor difference in protein loading).
To compare the fractional expressions from whole kidney among the three groups, the labeling density was corrected for difference in total amount of protein in each kidney by multiplication of the density by the total protein content.
Primary Antibodies
For semiquantitative immunoblotting, we used previously characterized monoclonal and polyclonal antibodies as follows. For Na-K-ATPase, a monoclonal antibody against the 1-subunit of Na-K-ATPase has been characterized (22). For the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1, LL320AP), an affinity-purified polyclonal antibody to the apical Na-K-2Cl cotransporter of the thick ascending limb has been characterized (16, 23). For AQP1 (CHIP serum or LL266AP), immune serum or an affinity-purified antibody to AQP1 has been characterized (48). For AQP2 (LL127 serum or LL127AP), immune serum or an affinity-purified antibody to AQP2 has been described (14). For AQP3 (LL178AP), an affinity-purified polyclonal antibody to AQP3 has been characterized (15).
Calculations and Statistics
GFR was estimated by calculating the renal clearance of 51Cr-EDTA (ClEDTA) as follows
![]() |
Filtration fraction (FF) was calculated as follows
![]() |
Filtered load of sodium (FLNa) was calculated as follows
![]() |
Excretion rate of sodium (UNaV) was calculated as follows
![]() |
Fractional excretion of sodium (FENa) was calculated as follows
![]() |
Solute free water reabsorption (TcH2O) was calculated as follows
![]() |
Values are means ± SE. One-way analysis of variance was performed for statistical analysis. If there was a significant difference, Bonferroni's test was performed to ascertain the difference between the groups. P < 0.05 was considered to be statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
After neonatally induced PUUO, RBF of the obstructed kidney progressively decreased. RBF in the obstructed kidney was reduced to 33% of the sham level at 5 wk, 50% at 12 wk, and 49% at 24 wk (P < 0.05; Fig. 1A, Table 1). Consistent with the progressive RBF reduction, there was a significant reduction in GFR after 24 wk of obstruction to 43% of sham levels (172 ± 36 vs. 306 ± 42 µl·min1·100 g body wt1, P < 0.05; Table 2). This progressive reduction in RBF was partially prevented in PUUO + 1wR rats. Examination of the rats at week 5 demonstrated that RBF was significantly higher in PUUO + 1wR than in PUUO rats (2.79 ± 0.54 vs. 1.64 ± 0.17 ml·min1·100 g body wt1, P < 0.05), whereas in PUUO + 4wR rats, RBF did not differ significantly from levels in PUUO rats (1.78 ± 0.25 vs. 1.64 ± 0.17 ml·min1·100 g body wt1, P > 0.05; Fig. 1A). At 24 wk after onset of obstruction, RBF was significantly reduced in PUUO + 1wR rats compared with sham-operated controls (1.28 ± 0.17 vs. 1.79 ± 0.12 ml·min1·100 g body wt1, P < 0.05), but levels were higher than in PUUO rats (1.28 ± 0.17 vs. 0.92 ± 0.17 ml·min1·100 g body wt1, P < 0.05) and PUUO + 4wR rats (1.28 ± 0.17 vs. 0.74 ± 0.13 ml·min1·100 g body wt1, P < 0.05; Fig. 1A). Consistent with these RBF changes in the released kidneys, the GFR reduction was prevented in PUUO + 1wR rats (304 ± 18 and 306 ± 42 µl·min1·100 g body wt1 in PUUO + 1wR and sham, respectively, P > 0.05), whereas GFR in PUUO + 4wR rats did not differ significantly from that in PUUO rats (137 ± 34 vs. 172 ± 36 µl·min1·100 g body wt1, P > 0.05; Table 2).
|
|
|
In the contralateral nonobstructed kidney of rats with PUUO, there was significant compensatory increase in RBF at 5 wk [5.17 ± 0.78 vs. 2.49 ± 0.19 (sham) ml·min1·100 gbody wt1, P < 0.05; Fig. 1B, Table 1]. This compensatory increase in RBF persisted at 12 wk [3.12 ± 0.32 vs. 2.49 ± 0.19 (sham) ml·min1·100 g body wt1, P < 0.05] and 24 wk [2.20 ± 0.18 vs. 1.80 ± 0.10 (sham) ml·min1·100 g body wt1, P < 0.05]. However, in the nonobstructed kidney of PUUO + 4wR rats, a similar compensatory increase in RBF was demonstrated at 5, 12, and 24 wk of age (Fig. 1B, Table 1). This compensatory increase in RBF was attenuated in the nonobstructed kidney of PUUO + 1wR rats compared with PUUO + 4wR rats (Fig. 1B, Table 1). Although it was significantly elevated at 5 wk [4.77 ± 0.54 vs. 2.49 ± 0.19 (sham) ml·min1·100 g body wt1, P < 0.05], RBF normalized to sham levels at 12 wk (2.99 ± 0.26 vs. 2.49 ± 0.19 ml·min1·100 g body wt1, P > 0.05) and at 24 wk after onset of obstruction (2.09 ± 0.12 vs. 1.80 ± 0.10 ml·min1·100 g body wt1, P > 0.05).
In contrast to the RBF increase in the nonobstructed kidneys, no compensatory increase in GFR was observed at 24 wk after onset of obstruction. GFR in the nonobstructed kidney was identical in all four groups (Table 2).
Early Release of Obstruction Prevents Progression of Hydronephrosis
PUUO caused a pronounced increase in TKV of the obstructed kidneys in the PUUO group (Fig. 2A). This increase persisted at 5 wk of age [1.49 ± 0.19 vs. 0.50 ± 0.02 (sham) ml/100 g body wt, P < 0.05], 12 wk of age [0.90 ± 0.15 vs. 0.38 ± 0.01 (sham) ml/100 g body wt, P < 0.05], and 24 wk of age [0.98 ± 0.25 vs. 0.38 ± 0.01 (sham) ml/100 g body wt, P < 0.05], whereas the kidney wet weight of the obstructed kidney did not change significantly in the PUUO rats at 24 wk of age [0.34 ± 0.03 vs. 0.32 ± 0.01 (sham) g/100 g body wt, P > 0.05; Table 3 ]. On the contrary, whole kidney protein content was significantly reduced in the obstructed kidney of the PUUO rats, consistent with the development of a markedly progressive hydronephrosis and obstructive nephropathy (Table 3). In the PUUO + 4wR group, development of TKV in the obstructed kidney did not differ from that in the PUUO group (Fig. 2A). However, release of obstruction after 1 wk (PUUO + 1wR group) completely abolished the increase in TKV. At 5, 12, and 24 wk, TKV of the obstructed kidney was similar to TKV in sham-operated control rats (Fig. 2A). Furthermore, wet kidney weight of the obstructed kidney in the PUUO + 1wR group was slightly reduced, but total protein was identical to that in sham-operated control kidneys, consistent with prevention of the development of obstructive nephropathy (Table 3).
|
|
Early Release of Obstruction Attenuates Natriuresis From the Obstructed Kidney
Neonatal PUUO did not change plasma concentrations of sodium and osmolality, whereas the concentration of sodium was significantly increased in urine from the obstructed kidneys (53.1 ± 12.7, 44.3 ± 8.9, and 21.8 ± 4.9 µmol/ml in PUUO, PUUO + 4wR, and sham, respectively, P < 0.05; Table 4). Consistent with the reduced GFR in obstructed kidneys, the filtered load of sodium decreased in the obstructed kidney in PUUO and PUUO + 4wR rats (Table 4). Furthermore, the fractional excretion of sodium [1.86 ± 0.62 vs. 0.37 ± 0.13% (sham), P < 0.05] and the urinary sodium excretion [0.32 ± 0.07 vs. 0.11 ± 0.02 (sham) µmol·min1·100 g body wt1, P < 0.05; Table 4] were significantly increased in the obstructed kidney in PUUO and PUUO + 4wR rats 24 wk after onset of obstruction (Table 4).
|
Early Release of Obstruction Normalizes Urinary Concentrating Capacity in the Obstructed Kidney
Neonatal PUUO did not change the total urine output. Consistent with the known impairment in renal water handling during obstructive nephropathy, TcH2O was markedly decreased in the obstructed kidney from PUUO rats (0.47 ± 0.16 and 2.71 ± 0.67 µl·min1·100 g body wt1 in PUUO and sham, respectively, P < 0.05; Table 4), demonstrating a reduced ability of these kidneys to reabsorb water in the collecting duct. This was slightly increased in the obstructed kidney in PUUO + 4wR rats (1.59 ± 1.02 µl·min1·100 g body wt1), whereas release of the obstruction after 1 wk demonstrated that TcH2O increased significantly compared with PUUO (1.73 ± 0.60 and 0.47 ± 0.16 µl·min1·100 g body wt1 in PUUO + 1wR and PUUO, respectively, P < 0.05; Table 4), suggesting that early release of obstruction reduced the urinary concentrating defect.
Early Release of Obstruction Prevents the PUUO-Induced Downregulation of Renal Sodium Transporters
To investigate the molecular mechanism involved in the defective sodium reabsorption in the obstructed kidney after neonatal PUUO, expression of the key sodium transporters BSC-1 and Na-K-ATPase was examined in whole kidney samples.
After 24 wk of persistent PUUO, semiquantitative immunoblotting demonstrated a reduced abundance of the 1-subunit of the Na-K-ATPase (62 ± 7 and 100 ± 11% in PUUO and sham, respectively, P < 0.05; Fig. 3, A and B, Table 5). BSC-1, another key sodium transporter responsible for the secondary active transport of NaCl in the medullary thick ascending limb, was not significantly changed in the obstructed kidney (112 ± 10 and 100 ± 28% in PUUO and sham, respectively, P > 0.05; Fig. 3, C and D, Table 5).
|
|
|
Early Release of Obstruction Prevents the PUUO-Induced Downregulation of Renal AQPs
Acute UUO is associated with a marked reduction in the expression of renal AQPs (18, 28), concurrent with the development of a urinary concentrating defect. To investigate the molecular mechanism involved in the impaired renal water handling in response to neonatal PUUO, expression of AQP1, located at the proximal tubule and descending thin limb of Henle, and the water channels AQP2 and AQP3, located in the collecting duct, was studied using immunoblot analysis. Semiquantitative immunoblotting using membrane fractions prepared from whole kidney revealed that a 24-wk period of obstruction was associated with markedly reduced AQP1 expression (Fig. 5, A and B, Table 5). Both AQP1 bands (29 and 3550 kDa) were decreased proportionally. Densitometric analysis revealed a significant decrease in AQP1 expression in PUUO rats (53 ± 3 and 100 ± 10% in PUUO and sham, respectively, P < 0.05).
|
Release of obstruction after 1 wk prevented the decreased expression of AQP1 (73 ± 12 and 100 ± 10% in PUUO + 1wR and sham, respectively, P > 0.05) and AQP3 (97 ± 9 and 100 ± 11% in PUUO + 1wR and sham, respectively, P > 0.05), consistent with the increased TcH2O. Release of obstruction after 4 wk revealed that obstructed whole kidney abundance of AQP1 did not differ significantly from the level in PUUO rats (Fig. 6, A and B), whereas obstructed whole kidney abundance of AQP2 was marginally increased compared with the level in the obstructed kidney from PUUO rats (Fig. 6, C and D).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Early Release of Obstruction Prevents RBF and GFR Reductions in the Obstructed Kidneys
Congenital urinary tract obstruction may cause profound changes in RBF and GFR of the obstructed kidney. This study showed that neonatal PUUO progressively reduced RBF, consistent with previous rat studies using the same model (51). This progressive reduction in ipsilateral RBF is a key manifestation of the development of obstructive nephropathy in response to congenital urinary tract obstruction as demonstrated by others (2, 4, 34). Development of the renal vasculature is delayed by neonatal UUO, and the activity of the intrarenal renin-angiotensin system is enhanced throughout the period of obstruction (4, 6, 9). Previous studies have also demonstrated that blockade of the renin-angiotensin system prevents some of the functional changes associated with neonatal obstruction of the ureter (4, 6, 13).
In this study, GFR was severely reduced by 40% at 24 wk after onset of neonatal PUUO. This finding is consistent with previous studies in guinea pigs in which chronic severe PUUO reduced GFR to a similar degree (7). During neonatal UUO, glomerular maturation is also delayed, and this may play an important pathophysiological role and in part explain the reduction in GFR. This is compatible with observations in the human fetus with obstructive nephropathy, which is associated with reduced numbers of glomeruli (19).
In rats with release of obstruction after 1 wk, GFR was normalized at 24 wk of age, whereas the reduction in RBF was significantly attenuated compared with RBF in the nonreleased kidney of PUUO rats. In contrast, in rats with release of obstruction after 4 wk, reduction in GFR and RBF was similar to the levels observed in nonreleased kidneys of PUUO rats. This demonstrates that early release of the obstruction on completion of nephrogenesis prevents the reduction in GFR. Previous studies have highlighted the significance of the irreversible nature of nephron loss resulting from chronic UUO in the developing kidney (5, 11, 17). This study demonstrated that the filtration fraction was identical and slightly elevated in all obstructed kidneys, whether obstruction was released after 1 wk or 4 wk or was not released. Thus, despite normalization of GFR, the finding that filtration fraction is not changed may indicate ongoing hyperfiltration in the obstructed kidneys, which could be due to a reduction in the total number of glomeruli. The primary concern is that PUUO associated with impaired nephrogenesis and hyperfiltration over time may lead to progressive glomerular sclerosis, which is more marked in immature than in adult rats subjected to unilateral nephrectomy 511 mo previously (40).
PUUO was associated with a significant compensatory increase in the contralateral RBF that persisted during the 24 wk of observation. This is consistent with the results of the previous studies demonstrating compensatory changes in RBF of the intact kidney in response to neonatal UUO (50). An important observation of this study was that the compensatory increase in the contralateral RBF was attenuated in rats with release of obstruction after 1 wk, suggesting a blunting of the counterbalance in this group compared with late release and no release of obstruction.
Early Release of Obstruction Prevents Development of Hydronephrosis
Neonatal PUUO was associated with development of severe hydronephrosis and obstructive nephropathy. This study showed that PUUO resulted in a dramatic increase in TKV, consistent with previous findings demonstrating a similar increase in TKV in response to severe neonatal PUUO (50, 51). Furthermore, weight of the obstructed kidney did not increase significantly, suggesting that the volume increase was due to an increased amount of water. In accordance with this, the renal protein content was reduced, consistent with obstructive nephropathy. Similar results were found in the rats with release of obstruction after 4 wk. In contrast, rats with release of obstruction after 1 wk merely manifested mild pelvic dilation at 24 wk of age, and renal protein content was similar to that in sham-operated controls, indicating that early release may prevent progressive structural damage of the renal parenchyma. These results also corresponded with previous findings demonstrating a significant renal atrophy in the postobstructed kidney in rats subjected to even 2 days of neonatally induced complete UUO (12). In this study, release of obstruction at 4 wk did not alter the pattern of RBF, TKV, and GFR changes induced by the neonatal PUUO in the obstructed or nonobstructed kidney. These results support the view that progressive damage of the obstructed kidney is more severe during the period immediately after nephrogenesis when renal maturation takes place than in the late phase of nephrogenesis (11). Thus this study indicates that timing of surgical intervention plays a key role in the outcome of renal function in response to neonatally induced ureteral obstruction. This study supports the previous clinical view that early surgical intervention provides the highest degree of preservation of renal function (41, 42).
After neonatal UUO, growth of the obstructed kidney is impaired. This study showed that although true growth was impaired by obstruction, as demonstrated by a reduction in protein content, a compensatory growth of the contralateral kidney occurred. This is consistent with previous data demonstrating compensatory growth in response to neonatal UUO and is related directly to the duration of obstruction (12, 50). This so-called counterbalance has also been found to be significantly greater in neonatal than in adult animals (46). Consistent with previous observations, this study demonstrated that volume of the contralateral nonobstructed kidney increased persistently during the 24 wk of obstruction. Renal counterbalance was not stimulated in PUUO + 1wR rats, further supporting the view that early release of obstruction protects the kidney from progressive damage.
Although a compensatory increase in kidney mass and RBF was demonstrated in the nonobstructed kidney, there was no compensatory increase in GFR. Indeed, total GFR was decreased. This observation suggests that compensation is not induced exclusively by the functional demands. Various mechanisms may be involved in the functional cross talk between the kidneys. A 1-yr observation study showed that contralateral GFR is not increased in rats subjected to 5 days of neonatal complete UUO (10). The lack of compensatory increase was associated with progressive tubular damage in the nonobstructed kidney, indicating that not only the obstructed kidney, but also the nonobstructed kidney, suffered during severe unilateral obstruction. Our study confirms that severe obstruction early in life is not accompanied by an adaptive increase in function.
Early Release of Obstruction Attenuates PUUO-Induced Natriuresis and Downregulation of Renal Sodium Transporters
Neonatal UUO has profound effects on the developing tubule, with suppression of proliferation and maintenance of an immature phenotype by tubular epithelial cells (3). To examine whether the suppressed maturation and the pronounced changes in renal hemodynamics and tubular function are associated with molecular changes of the tubular cells, the expression of various renal transporters was examined. An intact urine concentration is critically dependent on the hypertonic medullar interstitium, which is generated by active NaCl reabsorption as a consequence of countercurrent multiplication and the osmotic equilibration of water across the tubular epithelium via AQPs (24). The active transport of sodium occurs mainly via the key sodium transporters: the basolateral Na-K-ATPase (22), the type 3 Na/H exchanger (NHE3) (1), and the apical BSC-1 (or NKCC2) (16). This study demonstrated that Na-K-ATPase abundance in the obstructed kidney was decreased after 24 wk of obstruction. There was a defective reabsorption of sodium in the obstructed kidney, which was evidenced by the increase in sodium excretion. Thus it is likely that the reduced abundance of Na-K-ATPase plays a significant role in the increased urinary excretion of sodium from the obstructed kidney in PUUO rats. The present results support the view that renal sodium transport is critically affected by ureteral obstruction as previously demonstrated (30) and underscore the role of an intact expression of renal sodium transporters in maintaining an intact renal epithelial sodium transport in response to neonatal PUUO. Downregulation of Na-K-ATPase was prevented by release of the obstruction after 1 wk, demonstrating at the molecular level that early release of obstruction is important to protect the developing tubule system from damage. Release of obstruction after 1 wk also attenuated the natriuresis from the released kidney, demonstrating a functional association between the abundance of Na-K-ATPase and epithelial sodium transport. In contrast, release of obstruction after 4 wk demonstrated that abundance of Na-K-ATPase did not change compared with PUUO. This result further underscores that, in this system with severe neonatal PUUO, early release of the obstruction is essential to maintain the normal reabsorptive capacity of sodium.
The Na-K-ATPase is distributed along all nephron segments and functions in basolateral transport of sodium in the kidney tubule (22). This protein is involved in establishing the driving force promoting sodium reabsorption in the kidney tubule (22). The mechanisms involved in downregulation of the Na-K-ATPase are not fully understood. The reduced GFR and associated reduction in the filtered load of sodium may directly regulate the expression of Na-K-ATPase. Alternatively, the progressive hydronephrosis and development of obstructive nephropathy due to the direct effects of the increased interstitial pressure may also be important factors in the dysregulation of Na-K-ATPase. Because early release of obstruction was associated with prevention of the GFR decrease as well as progression of hydronephrosis, this study cannot explain which factor(s) is of most significance.
Early Release of Obstruction Prevents PUUO-Induced Downregulation of Renal AQPs
To examine whether dysregulation of renal AQPs is involved in the impaired urinary concentrating capacity, expression of the proximal nephron water channel AQP1 and the collecting duct water channels AQP2 and AQP3 was measured. AQP1, expressed in the proximal tubule and descending thin limb of Henle's loop, was significantly reduced in the obstructed kidney of PUUO and PUUO + 4wR rats. This finding is consistent with studies demonstrating that ureteral obstruction downregulates AQP1 (28, 29). It was recently demonstrated that AQP1 knockout mice manifest a severe urinary concentrating defect and a decreased transepithelial water permeability, indicating that AQP1 plays a pivotal role in the countercurrent multiplier system (31). Impairment of fluid reabsorption in the proximal tubule and descending thin limb may have significant effects on the urinary concentrating mechanism. Thus downregulation of AQP1 expression in this study may indicate that neonatal PUUO directly impairs water reabsorption in the proximal tubule and descending thin limb. As suggested previously (45), an altered regulation of proximal tubule function and GFR may be a result of resetting of the tubuloglomerular feedback response induced by an increased delivery of NaCl at the macula densa.
Consistent with recent studies, semiquantitative immunoblotting of the obstructed kidneys demonstrated that the protein expression of AQP2 was marginally reduced and AQP3 was significantly reduced in response to neonatal PUUO (18, 28, 29). In parallel, neonatal PUUO was associated with a severe reduction in solute free water reabsorption, demonstrating a functional association between AQP downregulation and water reabsorption at the collecting duct level.
The mechanisms responsible for the downregulation of collecting duct AQPs in response to obstruction remain unclear. It is well established that AQP2 is regulated, in the short and long term, by vasopressin (27, 37). Recent studies from our laboratory have provided evidence for vasopressin-independent regulation of AQP2 and AQP3 in response to acute obstruction in adult rats (28, 29).
An important finding of this study demonstrated that release of obstruction after 1 wk prevented downregulation of AQP2 and AQP3. Furthermore, the functional significance of this finding was demonstrated as a partial prevention of the reduction in solute free water reabsorption. Thus progressive obstruction with a sustained interstitial pressure may significantly dysregulate renal AQPs as a direct consequence of the increased interstitial pressure or an indirect induction of molecular changes in the tubule cells mediated through altered levels of hormone concentration.
Conclusion
In summary, this study confirmed that neonatal PUUO in rats is associated with marked long-term changes in RBF and glomerular and tubular functions. The changes in tubular function could in part be explained by dysregulation of renal sodium transporters and water channels, suggesting that impaired renal handling of sodium and water in response to neonatal PUUO may be a direct consequence of this dysregulation. Importantly, this study demonstrated that release of obstruction after 1 wk, but not after 4 wk, prevented the majority of the changes in kidney function induced by PUUO and thus protected the kidney from obstructive damage. In conclusion, this study demonstrates that early release of neonatal obstruction before the fast maturation period following completion of nephrogenesis provides a better protection of renal functions than release of obstruction after the maturation process is completed.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|