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【关键词】 vasopressin
Departments of Medicine and Pediatrics, University of Colorado School of Medicine, Denver, Colorado
Division of Critical Care Nephrology, Chang Gung Memorial Hospital, Taipei, Taiwan
ABSTRACT
The purpose of this study was to examine protein expression of renal aquaporins (AQP) and ion transporters in hypothyroid (HT) rats in response to an oral water load compared with controls (CTL) and HT rats replaced with L-thyroxine (HT+T). Hypothyroidism was induced by aminotriazole administration for 10 wk. Body weight, water intake, urine output, solute and urea excretion, and serum and urine osmolality were comparable among the three groups at the conclusion of the 10-wk treatment period. One hour after oral gavage of water (50 ml/kg body wt), HT rats demonstrated significantly less water excretion, higher minimal urinary osmolality, and decreased serum osmolality compared with CTL and HT+T rats. Despite the hyposmolality, plasma vasopressin concentration was elevated in HT rats. These findings in HT rats were associated with an increase in protein abundance of renal cortex AQP1 and inner medulla AQP2. AQP3, AQP4, and the Na-K-2Cl cotransporter were also increased. Moreover, 1 h following the oral water load, HT rats demonstrated a significant increase in the membrane-to-vesicle fraction of AQP2 by Western blot analysis. The defect in urinary dilution in HT rats was reversed by the V2 vasopressin antagonist OPC-31260. In conclusion, impaired urinary dilution in HT rats is primarily compatible with the nonosmotic release of vasopressin and increased protein expression of renal AQP2. The impairment of maximal solute-free water excretion in HT rats, however, appears also to involve diminished distal fluid delivery.
water transport; solute-free water excretion; thyroid disease
HYPOTHYROIDISM IS A COMMON clinical disorder that is associated with significant abnormalities in systemic and renal hemodynamics and renal handling of salt and water. Patients and experimental animals with hypothyroidism demonstrate impaired water excretion, failure to achieve maximal urinary dilution, and hyponatremia (4, 6, 15, 20). Maximal solute-free water excretion is dependent on factors including 1) appropriate delivery of fluid to the diluting segments of the distal nephron, determined by glomerular filtration rate and proximal tubular fluid and sodium reabsorption; 2) ion reabsorption in water-impermeable segments of the nephron; 3) decreased secretion of arginine vasopressin (AVP) in response to serum hyposmolality; and 4) decreased collecting duct solute-free water reabsorption via aquaporin (AQP)-2 water channels in response to suppressed plasma AVP concentrations. Previous studies have suggested that dysregulation of each of these factors contributes to impaired urinary dilution in hypothyroidism (4, 6, 15, 20). However, a comprehensive molecular analysis has not been undertaken. The goal of this study was to examine the molecular mechanisms contributing to impaired urinary dilution in hypothyroidism.
METHODS
Animal model. The study protocol was approved by the University of Colorado Institutional Animal Care and Use Committee. Animals were housed individually in metabolic cages (Nalgene metabolic cages; Nalge, Rochester, NY) and were subjected to 12:12-h light-dark cycles and a constant ambient temperature. These cages provide separation of the urine and feces using the combination of a collecting duct funnel and a separating cone in the lower chamber. All animals underwent acclimation to the metabolic cages for a continuous 5-day period before initiation of the study. Male Sprague-Dawley rats weighing 260300 g were used for the experiments. Forty-eight rats were divided into three study groups: hypothyroid (HT; n = 16), hypothyroid with thyroxine replacement (HT+T; n = 16), and control (CTL; n = 16). All rats were subjected to the study protocol as outlined below.
Hypothyroidism was induced by administration of aminotriazole (Sigma, St. Louis, MO) for 10 wk (0.5 g/kg powdered rat chow) and was confirmed by tail vein serum total thyroxine measurement. HT+T rats received the same aminotriazole treatment for 12 wk. However, in the last 2 wk of study, HT+T rats also received intraperitoneal injection of L-thyroxine (50 μg/kg, Sigma) every other day. The period of aminotriazole treatment was extended compared with our previous studies due to prior reports suggesting that a urinary diluting defect is more apparent in advanced hypothyroidism (20). Control rats received plain powdered rat chow. Food intake was controlled through daily administration of 15 g powdered chow per rat to control solute, protein, and caloric intake for the duration of the study. Powdered rat chow was obtained from Harlan Teklad Bioproducts (Indianapolis, IN) and contained 0.4% sodium. Drinking water was provided ad libitum. Water and food intake and urine output were monitored at least daily. Body weight was assessed at least weekly.
Toward the conclusion of the aminotriazole treatment period, echocardiography was performed in nine rats in each of the three study groups using a GE Vingmed System Five imaging tool for small rodents with a 10-MHz probe (2). The animals were anesthetized with ketamine (40 mg/kg body wt ip) and xylazine (5 mg/kg body wt ip). The left ventricular fraction of shortening was calculated from left ventricular systolic (LVIDs) and diastolic (LVIDd) diameter as {[(LVIDd - LVIDs)/LVIDd] x 100%}. Cardiac output was calculated via measurement of the diameter of the outflow tract (LVOT), the flow through the outflow tract (VTI), and the heart rate (HR) by the formula 0.785 x LVOT2 x VTI x HR. The cardiac index was reported as cardiac output per 100 g of body weight. Animals were allowed to recover from the procedure and then were returned to metabolic cages.
Two to three days following echocardiography, a 24-h water balance study was performed. Urine was collected under oil and frozen for later analysis of osmolality and creatinine. Tail vein blood sampling was performed at the conclusion of the balance study to measure serum osmolality, creatinine, and free thyroxine concentration.
Subsequently, food and water were removed for 3 h. Thirty rats (i.e., HT, n = 10; HT+T, n = 10; CTL, n = 10) were given an oral water gavage of 50 ml/kg and were returned to the metabolic cages. Each urine sample spontaneously voided over the next 1-h period was collected, the volume was recorded, and osmolality was assessed. The percent water load excreted was determined as the volume of urine excreted in 1 h divided by the volume of water given by gavage x 100%. At 1 h following the oral water gavage, the rats were killed by decapitation to avoid any influence of anesthesia on plasma AVP concentration (9). Trunk blood was collected for plasma AVP concentration, serum sodium concentration, serum osmolality, and serum creatinine concentration. The left kidney was weighed. Tissue was prepared for examination of renal ion and urea transporters and AQP water channels as described below. Tissue from seven rats in each group was utilized for Western immunoblot studies, whereas tissue from three rats in each group was utilized for immunohistochemistry.
In six additional rats in each of the three study groups, food and water were removed for 3 h. These rats were then given the nonpeptide vasopressin V2 receptor antagonist 5-dimethylamino-1-[4-(2-methylbenzoylamino)-benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine hydrochloride (OPC-31260; Otsuka Pharmaceutical, Tokyo, Japan) by oral gavage at a dose of 30 mg/kg (+OPC) (23). One hour following administration of OPC-31260, rats were given an oral water gavage of 50 ml/kg and were returned to metabolic cages. Each urine sample spontaneously voided over the next 1-h period was collected, the volume was recorded, and osmolality was assessed. The percent water load excreted was determined as described above. Urine flow rate and minimal urine osmolality for the 1-h time period following oral water gavage were assessed and recorded.
Protein isolation. For 21 rats (HT, n = 7; HT+T, n = 7; CTL, n = 7), following decapitation, one kidney from each rat was placed in ice-cold isolation solution containing 250 mM sucrose, 25 mM imidazole, and 1 mM EDTA, pH 7.2, with 0.1% vol protease inhibitors (0.7 μg/ml pepstatin, 0.5 μg/ml leupeptin, 1 μg/ml aprotinin) and 200 μM phenylmethylsulfonyl fluoride. Kidneys were then dissected on ice into cortical, outer medullary, and inner medullary regions. Tissue samples were immediately homogenized in a glass homogenizer at 4°C. After homogenization, protein concentration was determined for each sample by the Bradford method (Bio-Rad, Richmond, CA).
Western blot analysis. Western blot analysis was performed to examine kidney expression of renal cortex AQP1, sodium-potassium-2 chloride cotransporter (Na-K-2Cl), sodium-potassium-ATPase (Na-K-ATPase), and sodium-hydrogen exchanger (NHE)-3; outer medulla Na-K-2Cl cotransporter; and inner medulla AQP2, AQP3, and AQP4. SDS-PAGE was performed on 8 or 12% gels. After transfer by electroelution to polyvinylidene difluoride membrane (Millipore, Bedford, MA), blots were blocked overnight with 5% nonfat dry milk in PBS() and then probed with the respective antibodies for 24 h at 4°C. After being washed with buffer containing PBS() with 0.1% Tween 20 (J. T. Baker, Phillipsburg, NJ), the membranes were exposed to secondary antibody for 1.5 h at room temperature. Subsequent detection of the specific proteins was carried out by enhanced chemiluminescence (Amersham, Arlington Heights, IL) according to the manufacturer's instructions. Prestained protein markers were used for molecular mass determinations. Densitometric results were reported as integrated values (area x density of band) and expressed as a percentage compared with the mean value in controls (100%). For AQP2, immunoblots were assessed for multiple protein amounts, including 2, 1, and 0.5 μg/lane. This was done to ensure that blot quantitation was within the linear range for AQP2 quantitation. This was indeed the case. Densitometric measurement of both glycosylated and nonglycosylated bands for AQP2 and -3 was performed.
Membranes were stained with Coomassie blue to ensure equal loading. Western immunoblots as shown in RESULTS are representative of results obtained from all samples (HT, n = 7; HT+T, n = 7; CTL, n = 7). Densitometry results shown are means ± SE obtained from all samples.
Antibodies. Antibodies to AQP2, AQP3, AQP4, the Na-K-2Cl cotransporter, and NHE3 have been previously characterized (1, 5, 7, 21, 22). Anti-Na-K-ATPase 1 and 1 antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-AQP1 antibody was obtained from Chemicon International (Temecula, CA).
Differential centrifugation for AQP2 trafficking. Differential centrifugation was performed on inner medullary homogenates as previously described (5, 14). Once the above-mentioned Western blot analysis had been performed, inner medullary homogenates were initially centrifuged at 17,000 g for 20 min at 4°C to remove incompletely homogenized fragments and nuclei. The supernatant was saved. The pellets were then resuspended in ice-cold isolation solution with protease inhibitors, homogenized on ice twice for 15 s each, and then centrifuged again at 17,000 g for 20 min at 4°C. The pellets ("plasma membrane-enriched fraction") were retained, and the supernatants were combined and pelleted by centrifugation at 200,000 g for 60 min at 4°C ("intracellular vesicle-enriched fraction"). Protein measurement, sample preparation, and Western immunoblotting for AQP2 were performed as described above.
Biochemical measurements. Plasma AVP concentration was assessed by radioimmunoassay as described previously (10). Serum and urine osmolality was measured by freezing-point depression (Advanced Instruments, Norwood, MA). Serum and urine creatinine were measured (Beckman Instruments, Fullerton, CA). Twenty-four-hour creatinine clearance was used as an estimate of glomerular filtration rate.
Statistical methods. Statistical analysis of results was performed using analysis of variance with Tukey's post hoc test. Results are expressed as means ± SE, with P < 0.05 considered significant.
RESULTS
Physiological parameters before oral water loading. Serum-free thyroxine measurement confirmed successful induction of hypothyroidism in HT rats. Body weight was comparable in all study groups at the initiation and conclusion of the study (Table 1). This was anticipated, as all rats received the same intake of powdered rat chow. Consistent with our previous studies in short-term hypothyroidism (2), no significant differences were noted in water intake, urine output, urine osmolality, dietary sodium intake, serum osmolality, serum creatinine concentration, 24-h creatinine clearance, or urea or osmolar clearance between the three study groups before oral water loading. Fractional excretion of sodium was significantly higher in HT than HT+T.
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Consistent with our prior studies (2), this degree of hypothyroidism was associated with marked reductions in HR, cardiac output, cardiac index, and left ventricular fraction of shortening (Table 2). Kidney weight was also decreased in HT rats (1.6 ± 0.1 vs. CTL 2.2 ± 0.1 vs. HT+T 2.2 ± 0.1 g, P < 0.05 HT vs. CTL and HT+T) compared with the other two study groups.
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Effect of an oral water load on urinary dilution in hypothyroidism. With oral water loading of 50 ml/kg body wt, HT rats excreted significantly less water in 1 h compared with controls (Fig. 1A). This finding was associated with a significantly lower urine flow rate (Fig. 1B) and higher minimal urinary osmolality (Fig. 1C) in HT rats within 1 h of water loading as well as significantly decreased serum osmolality (Fig. 2A). However, despite this hyposmolality in HT rats, plasma AVP concentration was not suppressed compared with CTL and HT+T rats (Fig. 2B). In fact, plasma AVP concentrations remained significantly elevated in HT rats compared with the other two study groups.
Effect of an oral water load on AQP1 and sodium transport in hypothyroidism. Hypothyroidism was associated with a significant increase in the protein abundance of renal cortex AQP1 (Fig. 3A) compared with CTL and HT+T rats. No significant differences were observed between the study groups for AQP1 protein abundance in outer medulla [CTL 100 ± 13 vs. HT 90 ± 10 vs. HT+T 90 ± 13% CTL mean, P not significant (pNS)] or inner medulla (CTL 100 ± 17 vs. HT 76 ± 19 vs. HT+T 82 ± 13% CTL mean, pNS). Protein abundance of both cortex and outer medulla Na-K-2Cl cotransporter was significantly increased in hypothyroidism (Fig. 3, B and C). There was no significant difference in the protein abundance of the 1-subunit of renal cortex Na-K-ATPase among the groups (data not shown). However, there was a marked decrease in Na-K-ATPase 1-subunit protein abundance in HT rats compared with the other study groups (Fig. 4A). Renal cortex NHE3 (Fig. 4B) was markedly decreased in HT and increased in HT+T rats compared with CTL. There was a significant correlation between serum T4 concentration and renal cortex NHE3 protein abundance in all study groups (Fig. 4C).
Effect of an oral water load on AQP2, 3, and 4 in hypothyroidism. In association with impaired urinary dilution, HT rats demonstrated increases in inner medulla AQP2 (Fig. 5A), AQP3 (Fig. 5B), and AQP4 (Fig. 5C) compared with both CTL and HT+T rats.
Effect of an oral water load on trafficking of AQP2. An increase in the membrane to vesicle fraction of AQP2 in response to an acute water load during hypothyroidism was observed by Western immunoblotting of membrane and vesicle fractions of inner medulla (Fig. 6).
Vasopressin V2 receptor antagonism abolished the urinary diluting defect in hypothyroid rats. Administration of OPC-31260 was associated with marked increases in percent water excretion and urine flow rate within 1 h following the oral water load in all three study groups. The percent excretion and urine flow rate were, however, still significantly less in the HT rats. However, the defect in urinary dilution, as assessed by minimal urinary osmolality, in the HT rats was corrected totally by the V2 vasopressin antagonism with OPC-31260 (Table 3).
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DISCUSSION
Impaired urinary dilution in association with hypothyroidism has been recognized for several decades (6, 15). However, only recently have the molecular tools become available to fully elucidate the many defects that contribute to this abnormality in water handling. Utilizing a model of chronic severe myxedema, we were able to confirm a hypothyroidism-associated impairment in urinary dilution and solute-free water excretion. It should be noted that our approach using matched feeding between study groups resulted in limitation of daily food intake for CTL and HT+T rats. Thus we cannot exclude the possibility that such limitation affected the described results. However, we believed that controlled solute intake among the three groups was of primary importance in this study. In our study, HT rats were unable to normally excrete an oral water load compared with CTL rats. Serum hyposmolality and a significant increase in minimal urinary osmolality occurred in association with retention of the oral water load in these HT animals.
Several processes are known to mediate urinary diluting ability in animals and humans, including delivery of sufficient fluid to the diluting segment of the distal nephron, ion reabsorption in water-impermeable nephron segments, and appropriate suppression of AVP-mediated AQP2 water reabsorption. In particular, it has been proposed that the marked decline in renal function associated with hypothyroidism may result in impaired distal delivery of fluid (4), thus limiting the amount of solute-free water available for excretion. In our model of advanced hypothyroidism, diminished cardiac output resulting in arterial underfilling may have been a stimulus for enhanced renal cortex AQP1 protein abundance. Despite the significant alterations in systemic hemodynamics, renal function, as assessed by serum creatinine concentration and creatinine clearance, was comparable between the study groups. Thus the impact of impaired glomerular filtration rate on distal fluid delivery was minimized. However, hypothyroidism was associated with a marked upregulation of renal cortex AQP1. AQP1 is known to be important in proximal tubular reabsorption of water (16), and deficiencies of this water channel are associated with impaired urinary concentration in both animals and humans (11, 13). The upregulation in AQP1 expression in the HT rats is compatible with increased proximal fluid reabsorption and diminished fluid delivery to the distal nephron diluting segments.
The HT rats also demonstrated an upregulation of the Na-K-2Cl cotransporter in the renal cortex and outer medulla. Intact reabsorption of sodium via this cotransporter in the water-impermeable thick ascending limb of the loop of Henle is critical to the generation of dilute urine. Enhanced immunostaining for Na-K-2Cl cotransporter has been described previously in hypothyroidism induced by methimazole (18). It is known that this transporter is regulated by vasopressin, and thus it seems likely that the elevated circulating AVP concentration observed in our study may contribute to this finding.
We found no significant differences in protein abundance of the 1-subunit of renal cortex Na-K-ATPase among the study groups. However, the HT rats demonstrated a significant decrease in the abundance of renal cortex Na-K-ATPase 1-subunit compared with CTL and HT+T rats. These findings are consistent with previously reported studies that have attributed decreased Na-K-ATPase activity in hypothyroidism to a selective decrement in the -subunit of Na-K-ATPase (8, 17). The protein abundance of renal cortex NHE3 was also diminished in HT rats, consistent with the known effect of T3 to stimulate NHE3 activity by activating NHE3 gene transcription and increasing NHE3 transcript and protein abundance (3). These alterations in -Na-K-ATPase and NHE3 protein abundance likely contribute to the increased fractional excretion of sodium observed in HT rats.
Finally, a major factor in the production of dilute urine is suppression of AVP-mediated AQP2 water reabsorption in the renal collecting duct. Previous investigators have suggested that this impairment in solute-free water formation in hypothyroidism relates to inappropriately high circulating levels of plasma AVP (19, 20). This was confirmed in our study, as HT rats demonstrated a significant increase in plasma AVP concentration 1 h following the oral water load compared with either CTL or HT+T rats. This is particularly remarkable because the observed significant decrease in serum osmolality should have been sufficient to osmotically suppress plasma AVP concentration in HT rats. Therefore, it appears that the nonosmotic release of AVP is an important factor in impaired urinary dilution in this model of hypothyroidism. The reversal of the impaired urinary diluting ability, as assessed by minimal urinary osmolality, by administration of a vasopressin V2 receptor antagonist to HT animals further supports this conclusion. It is important to note, however, that maximal water excretion in the HT rats increased but was still significantly less than in the two control groups. This finding is compatible with diminished distal fluid delivery in the HT rats.
It was suggested over two decades ago that defective urinary dilution in the hypothyroid state was due to "increased back diffusion of water in the distal nephron" (15). Using sophisticated molecular techniques, we were able to confirm that this is indeed the case. Specifically, hypothyroidism was associated with an increased protein abundance of renal inner medullary water channels AQP2, -3, and -4. It is now well established that renal collecting duct water permeability via AQP2 can be regulated in both a short-term (minutes) and long-term (hours to days) manner (12). Increased protein abundance of AQP2 by immunoblotting in this condition indicated long-term upregulation of the protein in response to chronic endogenous vasopressin exposure. However, increased short-term regulation of AQP2 by vasopressin was also evident in hypothyroidism. As shown in Fig. 6, increased plasma membrane-to-vesicle ratios were increased in HT rats compared with the other study groups 1 h following a water load. This increase in membrane AQP2 relative to cystosolic AQP2 would be anticipated to diminish urinary diluting capacity in HT rats in response to an oral water load.
In summary, the impairment in urinary dilution associated with hypothyroidism involves several tubular defects. The predominant mechanism of the defect in urinary dilution involves the nonosmotic release of vasopressin with increased protein expression of renal AQP2. Diminished distal fluid delivery also appears to be involved in the impaired maximal water excretion in HT rats.
GRANTS
This work was supported by The National Institutes of Health (DK-19928). Y.-C. Chen was supported by a grant from the Chang Gung Memorial Hospital (Taipei, Taiwan).
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.
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