Short-Term Hypothyroidism and Vasopressin Gene Expression in the Rat

Short-Term Hypothyroidism and Vasopressin Gene Expression in the Rat Randy L. Howard, MD, Sandra Summer, BS, Noreen Rossi, MD, Jin K. Kim, PhD, and Robert W. Schrier, MD • Hypothyroidism is associated with abnormalities in renal water handling, which include a delay in excretion of an acute water load, decreased urinary concentrating ability, and increased urine volume. In the present study, we investigated the role of vasopressin in aminotriazole-induced hypothyroidism by measuring vasopressin concentration in the plasma and pituitary along with vasopressin mRNA levels in the hypothalamus. After 5 weeks of aminotriazole treatment, L-thyroxine levels were Significantly lower in the experimental animals (122 ± 8 v 26 ± 1 nmol/L [9.5 ± 0.6 v2.0 ± 0.1Itg/dL]; P < 0.001). Serum sodium (148 ± 0.5 v 144 ± 1.2 mmol/L [mEq/L]; P < 0.01), and plasma osmolality (311 ± 2.5 v 304 ± 1.8 mmol/kg [mOsm/kg] H20; P < 0.05) were also lower in the experimental animals. There were no differences in plasma (1.9 ± 0.4 v 1.5 ± 0.2 pg/mL) or pituitary (1.5 ± 0.4 v 1.5 ± 0.2 ltg/pituitary) vasopressin levels. In addition, steady-state vasopressin mRNA levels were not different between the two groups (1,286 ± 210 v 1,093 ± 138 pg/hypothalamus). One week of L-thyroxine replacement resulted in significant increases in serum thyroxine levels without changes in the other variables measured. These results indicate that short-term hypothyroidism, which has been shown to exert substantial effects on renal function, causes only a modest central alteration in She plasma vasopressin-osmolality relationship, which occurs in the absence of detectable changes in vasopressin synthesis. © 1992 by the National Kidney Foundation, Inc. INDEX WORDS: Hypothyroidism; plasma vasopressin; pituitary vasopressin; vasopressin gene expression.

H

YPOTHYROIDISM is associated with alterations in renal function in humans and experimental animals. There is a delay in excretion of an acutely administered water load in both hypothyroid patients and animals. 1·3 In addition, decreased urinary concentrating ability 4-9 and increased urine volume 9 . 12 have been reported in hypothyroid humans and animals. Hyponatremia may also occur in myxedema, although usually in only the most severe cases. Arginine vasopressin (A VP) has two sites of action in the kidney that might explain the abnormalities noted above. In the coUecting tubules, AVP has direct effects on water transport, while in the medullary thick ascending limb of Henle's loop, vasopressin has effects on sodium chloride reabsorption, which may increase the osmotic driving force for the hydrosmotic effect of vasopressin. Plasma vasopressin concentrations have been reported to be both elevated9 ,13,14 and normal 15- 17 in patients and experimental animals with hypothyroidism. A VPis synthesized in the hypothalamus and transported to the posterior pituitary for storage. Release from the posterior pituitary into the blood occurs in response to osmotic and nonosmotic (eg, hypovolemia, hypoxia, pain) stimuli. This release of vasopressin from the posterior pituitary can occur quickly and may

be effected by a variety of factors, such as the form of euthanasia used and handling of the experimental animal before it is killed. Sudden release of vasopressin into the blood may result in difficulty in interpretation of the data when plasma vasopressin levels are the only variable investigated. Previous studies have reported only plasma vasopressin concentrations in response to hypothyroidism. 9 , 14 To avoid these problems, we have made measurements at three sites of the vasopressin synthetic and secretory pathway (plasma vasopressin, pituitary vasopressin, and hypothalamic vasopressin mRNA concentrations) to determine effects on vasopressin metabolism. The present study was undertaken to determine if 5 weeks of hypothyroidism was associated with changes in serum sodium, plasma vasopressin, pituitary vasopressin, or hypothalamic vasopressin mRNA levels. In addition, the effect From the Departments o/Medicine, University o/Colorado Health Sciences Center, Denver, CO; and Wayne State University School 0/ Medicine, Detroit, MI. Supported by National Institutes o/Health Grants No. DK 07135 and DK 19928. Address reprint requests to Robert W. Schrier, MD, Chairman, Department o/Medicine, Universityo/ColoradoHealth Sciences Center, 4200 E 9th Ave, Box B-1 78, Denver, CO 80262. © 1992 by the National Kidney Foundation, Inc. 0272-6386/92/1906-0011$3.00/0

American Journal of Kidney Diseases, Vol XIX, No 6 (June), 1992: pp 573-577

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HOWARD ET AL

574

of various forms of euthanasia on plasma vasopressin levels was investigated. METHODS

Effect of Euthanasia on Plasma Vasopressin Concentration Eighteen male Sprague-Dawley rats weighing 200 to 250 g were used for the experiments. Animals were divided into three groups (control, intraperitoneal pentobarbital, CO2 narcosis) of six animals each. All animals were killed by decapitation using a guillotine and trunk blood was collected for plasma vasopressin determination. Control animals were allowed to walk voluntarily into a plastic restrainer and were killed when appropriately positioned in the guillotine. Other animals received either intraperitoneal pentobarbital (30 mg/ kg body weight) or were allowed to breathe 100% CO2 gas for 2 to 5 minutes. Animals in the latter two groups were killed when voluntary movement could no longer be elicited. Plasma vasopressin levels were determined using a modification of the method of Anderson et aI. 18 This method uses a sensitive radioimmunoassay previously developed in our laboratory. Vasopressin antiserum (no. 2849) was generously supplied by Jacques Durr, MD.

Effect of Hypothyroidism on Vasopressin Metabolism Male Sprague-Dawley rats (body weight, 225 to 265 g) were divided into three groups of 10 animals each (control, hypothyroid, hypothyroid + L-thyroxine). Control animals received standard rat chow and were not manipulated until the end of week 4 as described below. Hypothyroidism was induced by adding 3-amino-I,2,4-triazole (Sigma, St Louis, MO) to standard rat chow. 10 Animals were allowed ad libitum water intake. After 4 weeks, animals were placed under light methoxyflurane anesthesia (Pitman-Moore, Washington Crossing, NJ) and either carrier-binder or 5 mg L-thyroxine sustained release pellets (Innovative Research of America, Toledo, OH) were implanted subcutaneously. These pellets provide a 3week controlled dose-dependent release rate. Seven days after implantation of the pellets, animals were killed by decapitation without anesthesia. The whole hypothalamus and pituitary were quickly dissected and frozen on dry ice. Trunk blood was collected for vasopressin, creatinine, sodium, osmolality, and L-thyroxine determinations. Serum sodium was determined using a flame photometer (Instrumentation Laboratory, Lexington, MA), plasma osmolality was determined by freezing point depression (Advanced Instruments, Needham Heights, MA), and creatinine was determined using a Beckman creatinine analyzer 2 (Beckman Instruments, Fullerton, CAl. Serum L-thyroxine levels were determined by radioimmunoassay in the central laboratory of this medical center and plasma vasopressin concentrations were determined as described previously.18 Pituitary vasopressin levels were determined after the whole pituitary was homogenized in 1.0 mL of 0.1 N HCI using the above assay. Total RNA from the hypothalamus was extracted by the method ofChomczynski and Sacchi.19 The frozen hypothalamus was homogenized in a solution containing 4 moVL guanidinium isothiocyanate, 25 mol/L sodium citrate (pH 7),

0.5% sarcosyl, and 0.1 mol/L 2-mercaptoethanol. Sequentially, 0.05 mL of 2 mol/L sodium acetate (pH 4), 0.5 mL phenol, and 0.1 mL chloroform-isoamylaicohol were added to the homogenate with thorough mixing after each addition. The final suspension was shaken vigorously for 10 seconds and placed on ice for 15 minutes. Following centrifugation at 10,000 X g for 2 minutes, the aqueous phase was transferred to a fresh tube and I vol of isopropanol was added. The RNA was precipitated at - 20 c C overnight and then pelleted by centrifugation at 10,000 X g for 20 minutes. The RNA pellet was redissolved in 0.15 mL of the homogenizing solution and precipitated with I vol isopropanol at - 20 c C for 3 hours. The RNA was again pelleted by centrifugation, washed twice with 75% ethanol, vacuum-dried, and resuspended in Tris-EDTA buffer. Vasopressin mRNA was determined by solution hybridization 20 using 35S-labeled antisense (probe) and sense (standard) RNA produced by in vitro transcription of a rat cDNA specific for the vasopressin gene provided by Drs Schmale and Richter as previously described. 21 Five micrograJrn) total RNA was added to each hybridization reaction and each sample was hybridized in triplicate. Hybridization for the standard curve was performed from 62.5 to 500 pg and was linear throughout this range. Slot-blot hybridization of total cellular RNA samples to a random primer-labeled (Prime-a-Gene system, Promega, Madison, WI) mouse /3-actin probe was used as a control. Briefly, total cellular RNA was applied to nitrocellulose (Nitroplus-2000, Micron Separations, Westboro, MA) using a Schlicher and Schuell apparatus and immobilized by baking at 80 c C for 2 hours. Prehybridization and hybridization solutions consisted of 0.1% sodium dodecyl sulfate (SDS), 50% formamide, 5X SSC (IX ssc = 0.15 mol/L NaCi + 0.015 mol/L sodium citrate), IX Denhardt's, 50 mmol/L NaPO., and 250 Ilg/mL denatured salmon sperm DNA. Following prehybridization for 6 hours at 42 c C, hybridization was performed using I X 106 cpm/mL at 42 c C overnight. The membrane was washed to a stringency of 0.1 X SSC, 0.1 % SDS at 50 c e. Autoradiography using Kodak XAR film (Eastman Kodak, Rochester, NY) and densitometric scanning were then performed. Statistics were performed using one-way analysis of variance (ANOVA) or tests for nonparametric data as appropriate. P values less than 0.05 were considered significant.

RESULTS

Effect of Euthanasia on Plasma Vasopressin Plasma vasopressin concentrations for each euthanasia group are shown in Table 1. Using both the standard 200 JLL of sample and a further dilution using 100 JLL of sample, plasma vasopressin levels were above the upper limit of the assay (200 pg/mL) in all animals in the CO2 narcosis group. The plasma vasopressin levels in both the intraperitoneal pentobarbital and CO2 narcosis groups were significantly higher than in the control group.

575

HYPOTHYROIDISM AND VASOPRESSIN METABOLISM Table 1. Plasma Vasopressin Levels

No anesthesia Intraperitoneal pentobarbital CO2 narcosis

N

PlasmaAVP (pg/mL)

6

0.7 ± 0.07*

6 6

3.9 ± 0.97 >200

P Value v Control

<0.026 <0.001t

"Mean ± SEM. t Statistics performed using 200 pgjmL.

Effect of Hypothyroidism on Vasopressin Metabolism Hypothyroidism, as measured by serum Lthyroxine, was induced in those animals receiving 3-amino-I,2,4-triazole, and placement of the subcutaneous L-thyroxine pellets resulted in significantly higher serum L-thyroxine levels than in either the control or hypothyroid animals (Table 2). Weight gain and hematocrits are shown in Table 2. Serum sodium and plasma osmolality were decreased significantly in both the hypothyroid and L-thyroxine replacement groups when compared with controls (Table 2). One week of L-thyroxine replacement did not result in changes in the serum sodium or plasma osmolality when compared with the hypothyroid group (Table 2). There were no differences in the plasma vasopressin, pituitary vasopressin, or hypothalamic vasopressin mRNA levels between the groups (Table 3). There were no differences between the groups in the gene expression of {1-actin (data not shown). DISCUSSION

The hypothyroid state in humans and experimental animals is known to be associated with

abnormalities of the renal concentrating4-12 and diluting mechanism. 1-3,13-15 Several studies ofhypothyroidism have incriminated abnormalities in the tubular response to vasopressin/,9,12 while others have demonstrated tubular defects in hypothyroid Brattleboro rats in the absence of vasopressin. 2 Other investigators have demonstrated elevated plasma vasopressin concentrations relative to the plasma osmolality in the hypothyroid state. 9,13,14 However, the effect of hypothyroidism on hypothalamic vasopressin gene expression has not heretofore been studied. We have developed techniques recently whereby effects of disease states, such as cardiac failure 21 and central diabetes insipidus,22 on hypothalamic messenger RNA for vasopressin can be studied in individual rats. These techniques have been used in the present study to examine the effect of hypothyroidism on vasopressin gene expression in the rat. The model of hypothyroidism used in the present study was that of aminotriazole feeding; after 5 weeks, a cellular defect in the medullary ascending limb response to vasopressin has been demonstrated as the primary tubular defect accounting for the animal's increased urine output and decreased urine osmolality.9 This tubular defect is accompanied by a significant decrease in glomerular filtration rate (GFR). The present study is the first to examine in hypothyroid rats the plasma and pituitary vasopressin concentrations along with the hypothalamic vasopressin mRNA. In the present study, the serum L-thyroxine concentrations decreased significantly at 5 weeks from 122 to 26 mmol/L (9.5 to 2.0 J,tg/ dL); this hypothyroid state was accompanied by constancy of vasopressin mRNA, pituitary stores,

Table 2. Blood Determinations Control

Variable

Weight gain (g) Hematocrit (%) Serum sodium (mmoljL) Serum creatinine, ILmoljL (mgjdL) Plasma osmolality (mmoljkg) Serum L-thyroxine, nmoljL (lLgjdL)

" P < 0.05 v control. t P < 0.01 v control. :f: P < 0.001 v control. § P < 0.001 v hypothyroid.

114 ± 44 ± 148 ± 53 ± 311 ± 122 ±

9.0 0.3 0.50 4 (0.6 ± 0.05) 2.54 8 (9.5 ± 0.60)

Hypothyroid

39 ± 44 ± 144 ± 44 ± 304 ± 26 ±

5.0:f: 0.4 1.15t 3 (0.5 ± 0.03) 1.75" 1:f: (2.0 + 0.09)

L-thyroxine

45 45 144 44 300 167

± ± ± ± ± +

4.0:f: 0.5 0.47t 5 (0.5 ± 0.06) 1.23:f: 21§ (13.0 + 1.63)

HOWARD ET AL

576 Table 3. Vasopressin Protein and Gene Expression Measurements Variable

Control

Hypothyroid

l-thyroxine

Plasma vasopressin (pgjmL) Pituitary vasopressin (JLgjpituitary) Hypothalamic vasopressin mRNA (pgjhypothalamus)

1.9 ± 0.40 1.5 ± 0.38 1,286 ± 210

1.5 ± 0.23 1.5 ± 0.21 1,093 ± 138

2.0 ± 0.36 1.2 ± 0.22 1,314 ± 142

and plasma concentrations (Tables 2 and 3). These results therefore document that the earliest and most pronounced effects of hypothyroidism occur at the renal9 and not central nervous system level. However, it should be noted that the modest but significant decrease in plasma osmolality in the hypothyroid rats may denote a slight "resetting" of the osmostat for vasopressin release after 5 weeks of hypothyroidism. As compared with the present results obtained without anesthesia, our previous results using pentobarbital anesthesia for euthanasia in the same model suggest that hypothyroidism may sensitize the rat to the nonosmotic effects of anesthesia on vasopressin release. 9 The modest change in the relationship between plasma vasopressin and plasma osmolality in the present study was not reversible with 7 days of L-thyroxine. The reversibility of the central effects of thyroid deficiency on the plasma vasopressin-osmolality relationship must therefore need more prolonged replacement therapy. In contrast, however, it is worth emphasizing that the renal effects of this degree of hypothyroidism in the rat have been shown to be reversible within 7 days of L-thyroxine treatment. 9 The proposal that more long-term hypothyroidism is necessary to demonstrate more pro-

nounced central perturbations in vasopressin synthesis, storage, and release is compatible with the observation of increased plasma vasopressin concentrations after 5 months of hypothyroidism in the rat 14 and the occurrence of hyponatremia in association with myxedema. 4 • 13 However, with long-term hypothyroidism, secondary consequences including alterations in cardiac and renal functien , as well as alterations in blood volume, may mediate effects on vasopressin synthesis, storage, and release. Moreover, with long-term hypothyroidism it must be recognized that hyponatremia has been recently shown to downregulate vasopressin synthesis in the rat. 23 In summary, except for a modest resetting of the vasopressin-osmolality relationship, shortterm hypothyroidism (5 weeks of aminotriazole) does not alter pituitary vasopressin stores orhypothalamic vasopressin gene expression. Thus, taken together with our previous results in the same experimental model, short-term hypothyroidism in the rat seems to exert the most pronounced effects at the level of the kidney, including a decrease in GFR, and vasopressin resistance at the level of the medullary ascending limb. 9

REFERENCES I. Bradley SE, Frederic S, Coehlho lB, et al: The thyroid and kidney. Kidney Int 6:346-365, 1974 2. Emmanouel DS, Lindheimer MS, Katz AT: Mechanism of impaired water excretion in the hypothyroid rat. 1 Clin Invest 54:926-934, 1974 3. Allon M, Harrow A, Pasque CB, et al: Renal sodium and water handling in hypothyroid patients: The role of renal insufficiency. 1 Am Soc Nephrol 1:205-210, 1990 4. Bloomer HA, Canary JJ, Kyle LH: Renal concentrating ability in patients with myxedema. Metabolism 10:469-474, 1961 5. DiScala VA, Kinney Ml: Effects of myxedema on the renal diluting and concentrating mechanism. Am 1 Med 50: 325-335, 1971 6. Holmes EW, DiScala VA: Studies on the exaggerated

natriuretic response to a saline infusion in the hypothyroid rat. 1 Clin Invest 49:1224-1236, 1970 7. Michael UF, Barenberg RL, Chavez R, et al: Renal handling of sodium and water in the hypothyroid rat. 1Clin Invest 51 :1405-1412, 1972 8. Vaamonde CA, Michael UF, Oster JR, et al: Impaired renal concentrating ability in hypothyroid man. Nephron 17: 382-395, 1976 9. Kim lK, Summer SN, Schrier RW: Cellular action of arginine vasopressin in the isolated renal tubules of hypothyroid rats. Am 1 PhysioI253:FI04-FllO, 1987 10. Davis RG, Madsen KM, Freg!y MJ, et al: Kidney structure in hypothyroidism. Am 1 Pathol 113:41-49, 1983 II. Fregly MJ: Increased water exchange in rats treated with antithyroid drug. 1 Pharmacol Exp Ther 134:69-76, 1969 12. Harkcom TM, Kim lK, Palumbo PJ, et al: Modulatory effect of thyroid function on enzymes of the vasopressin-sen-

HYPOTHYROIDISM AND VASOPRESSIN METABOLISM

sitive adenosine 3', 5'-monophosphate system in renal medulla. Endocrinology 102:1475-1484, 1978 13. Skowsky WR, Kikuchi TA: The role of vasopressin in the impaired water excretion of myxedema. Am J Med 64: 613-621, 1978 14. Seif SM, Robinson AG, Zenser TV, et al: Neurohypophyseal peptides in hypothyroid rats: Plasma levels and kidney response. Metabolism 28:137-143,1979 15. Waters AK: Increased vasopressin excretion in patients with hypothyroidism. Acta Endocrinol 88:285-290, 1978 16. Koide Y, Oda K, Shimizu K, et al: Hyponatremia without inappropriate secretion of vasopressin in a case of myxedema coma. Endocrinology (Japan) 29:363-368, 1982 17. Macaron C, Famuyiwa 0: Hyponatremia of hypothyroidism: Appropriate suppression of antidiuretic hormone levels. Arch Intern Med 138:820-822, 1978

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18. Anderson RJ, Pluss RG, Berns AS, et al: Mechanism of effect of hypoxia on renal water excretion. J Clin Invest 62:769-777, 1978 19. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159, 1987 20. Ournam OM, Palmiter RO: A practical approach for quantitating specific mRNAs by solution hybridization. Anal Biochem 131:385-393, 1983 21. Kim JK, Michel JB, Soubrier F, et al: Arginine vasopressin gene expression in chronic cardiac failure in rats. Kidney Int 38:818-822, 1990 22. Kim JK, Soubrier F, Michel J, et al: Arginine vasopressin gene regulation in the homozygous Brattleboro rat. J Clin Invest 86:14-16, 1990 23. Robinson AG, Roberts MM, Evron WA, et al: Hyponatremia in rats induces downregulation of vasopressin synthesis. J Clin Invest 86:1023-1029, 1990