AMERICAN JOURNAL OF PHYSIOLOGY

Vol. 229, No. 5, November 1975. Printed in U.S.A.

Effect of parathyroid hormone secretion on sodium

reabsorption by the proximal tubule

EDWARD G. SCHNEIDER

Department of Physiology, Center for the Health Sciences, University of Tennessee, Memphis, Tennessee 38163

SCHNEIDER, EDWARD G. EJect of parathyroid hormone secretion on sodium reabsorption by the proximal tubule. Am. J. Physiol. 229(5) : 1170-l 173. 1975.-To determine if an increase in the en- dogenous secretion of parathyroid hormone could decrease sodium reabsorption by the proximal tubule, the ionized calcium con- centration of blood perfusing the parathyroid gland of eight uni- laterally thyroid parathyroidectomized dogs (TPTX) was reduced by infusion of an isotonic sodium citrate plus sodium chloride solution into the blood supply of the parathyroid gland. The fractional clearance of phosphate increased significantly (+9.3 k 2.8 ml/min per 100 ml GFR), while fractional sodium re- absorption by the proximal tubule decreased (- .06 h .02; P < .025). In seven normal control dogs that received isotonic sodium chloride infusion, neither fractional sodium reabsorption by the proximal tubule nor the fractional clearance of phosphate was significantly altered. In five bilaterally TPTX dogs that received a sodium citrate plus sodium chloride infusion, sodium reab- sorption by the proximal tubule was not significantly altered. There were no significant changes in glomerular filtration rate or renal plasma flow in any of these groups. The data demonstrate that alterations in endogenous parathyroid hormone secretion can play a significant role in the regulation of sodium reabsorption by the proximal tubule.

dog micropuncture; phosphate excretion; plasma ionized calcium

THE IMPORTANCE 0F HORMONAL REGULATION of anion re- absorption as a physiological regulator of isotonic reab- sorption by the proxirnal tubule has recently been suggested by Knox and Davis ( 12). The possible role of parathyroid hormone as a regulator of not only phosphate reabsorption but also sodium reabsorption by the proximal tubule has been supported by micropuncture studies from several laboratories which have shown that the infusion of exoge- nous bovine parathyroid hormone inhibits sodium reab- sorption by the proximal tubule (I, 17). At present, how- ever, no experimental evidence has been presented which demonstrates that selective increases in endogenous para- thyroid hormone secretion can affect sodium reabsorption by the proximal tubule.

The purpose of this study was to determine if a selective increase in the endogenous secretion of parathyroid hor- mone could produce an inhibition of sodium reabsorption by the proximal tubule of the dog.

METHODS

Three groups of experiments were performed: group 1, eight unilaterally thyroparathyroidectomized v-mw

dogs received an infusion of a sodium citrate, sodium chlo- ride solution into the blood supply of the remaining para- thyroid gland; group II, five acutely TPTX dogs received an infusion of a sodium citrate, sodium chloride solution; and group III, seven dogs with intact parathyroid glands received an infusion of a sodium chloride solution.

All dogs were fed a standard pellet diet providing ap- proximately 30 meq of sodium per day. The dogs were allowed free access to water; food was withheld on the day of the experiment. The dogs were anesthetized with pento- barbital (30 mg/kg), a tracheotomy was performed, and the dogs were prepared for clearance and micropuncture as previously described ( 18).

In group I, immediately following removal of the right parathyroid and thyroid glands, the left carotid artery was occluded beyond the origin of the thyroid-parathyroid artery. A 23-gauge needle connected to an infusion pump was inserted into the left carotid artery 5-8 cm below the origin of the thyroid-parathyroid artery, and a 0.5-ml/min infusion of 0.9 % NaCl was initiated. After three successive 15-min clearance periods had been obtained, the carotid arterial infusion was changed to a 5-mg sodium citrate per minute per kilogram body weight infusion made isotonic by the addition of sodium chloride and adjusted to pH 7.4. Sixty minutes later three additional 15-min clearance periods were performed.

In group II experiments the dogs were bilaterally thyroid parathyroidectomized 2 h before starting the initial clear- ance measurements. These dogs were then treated in an identical manner to group I except that the sodium citrate solution was infused into the left jugular vein. In group III experiments the dogs received an infusion of isotonic sodium chloride solution (0.5- 1 .O ml/min) throughout the experimental protocol.

During the clearance measurements, micropuncture samples were obtained from late proximal tubule segments using the recollection micropuncture technique. Late proxi- mal tubule segments were identified by the injection of small nonocclusive oil droplets as previously described for this laboratory (15). Tubule fluid samples were collected at a rate which was sufficient to collect all the volume flow at the puncture site and hold a column of stained castor oil distal to the puncture to prevent retrograde flow ( 18). The volume of the tubule fluid sample was measured with a micropipette which was calibrated with a radioactive tracer. The single-nephron filtration rate (snfr) was calcu-

lated from the expression snfr = V, X (TF/P)r, where V,

is the volume collected per minute and the (TF/P) rn is

1170

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PTH SECRETION AND PROXIMAL SODIUM REABSORPTION 1171

the ratio of inulin concentration in tubule fluid to that in plasma. The concentration of inulin in the tubule fluid was determined in duplicate by a microfluorometric method (19). Fractional reabsorption (FR) of sodium and water by the proximal tubule was calculated from the following expression : FR = 1 - (P/TF) Ina Blood samples were collected at the midpoint of the 15-min urine collection periods. Inulin in plasma and urine was measured by the anthrone method. The PAH concentration in plasma and urine was measured by the method of Harvey and Brothers (10). Renal plasma flow was calculated from the clearance and extraction of PAH. Sodium and potassium concentra- tions in plasma and urine were measured by flame pho- tometry. Phosphate in plasma and urine was measured by the method of Young (20). Calcium and magnesium in plasma and urine were measured by atomic-absorption spectroscopy.

In group I peripheral and carotid arterial blood for ionized calcium were collected into a syringe and then injected into S-ml Vacutainers (Becton-Dickinson, Rutherford, N. J.) containing 143 PU heparin. Plasma was withdrawn anaero- bically into a tuberculin syringe through the rubber stopper of the Vacutainer after contrifugation at 3,000 rpm for 10 min. For measurements of ionized calcium in plasma, the Orion flow-through calcium-activity electrode (16) was used with the following modifications: I> standards were prepared in Vacutainers containing the same amount of heparin as the samples. 2) No trypsin or triethanolamide was added to the standards, which were prepared weekly, and 3) the membrane was primed by pooled normal plasma before the daily standard curve was run. Plasma pH was measured by a microelectrode and a standard pH meter.

In three dogs in group I and two dogs in group 11, arterial blood was obtained during the control and experimental periods for measurement of serum immunoreactive para- thyroid hormone (iPTH) concentration. The techniques of measurement of serum iPTH were the same as those reported in a previous publication (2). The antiserum used in the present studies was termed CHl 2M (chicken anti- bovine PTH) and was described in the previous report. In the present studies, a crude saline extract of pooled normal dog parathyroid glands was used as a standard in all radioimmunoassays. This was assigned an arbitrary value of 1,000 Ill-eq/ml, and immunoassay curves produced with this standard were superimposed on curves produced with multiple dilutions of a serum obtained from a dog made chronically hypocalcemic with citrate infusions. 1311~

labeled bovine PTH was used in assays as the labeled hormone species. Antiserum CHlZM reacts with synthetic bovine PTH l-34 (Beckman Bio-products) almost as well as with bovine PTH l-84 and, therefore, recognizes the biologically active region of the PTH’molecule. Sera from normal dogs consistently decreased the ratio of antibody bound to free 1311-labeled bovine PTH (B/F ratio) by 30- 50 %, whereas sera from hypoparathyroid dogs did not alter this ratio significantly, indicating that iPTH was being measured and not some nonspecific effect of serum on the immune system. In order that this potential problem could be circumvented, we used hypoparathyroid dog serum in assays as a blank and made corrections for small nonspecific changes in the B/F ratio as has been described (12). All measurements of serum iPTH in the present

study were done in duplicate in three different serum dilutions. In tra-assay and in terassay variations were 12 and 15 %, respectively.

The average for a variable during the initial clearance periods was compared by the Student I test for paired comparison to the average obtained for that variable during the experimental clearance periods. Differences between groups were compared by the Students t test for group comparison. The data are expressed as the mean value zt 1 SE.

RESULTS

A summary of the plasma calcium and plasma phosphate data for all dogs is presented in Table 1. Following the in- fusion of a sodium citrate solution into the blood supply of the parathyroid gland (group I), arterial ionized calcium concentration obtained from the left carotid artery was significantly less ( - -88 + -31 mg/ 100 ml ; P < .05) than the ionized calcium concentration obtained from femoral arterial blood. The femoral arterial ionized calcium con- centration was not significantly changed (.OO =t .03 mg/ 100 ml) (Table 1) following this infusion. Total plasma calcium was not significantly changed in group I (+. 3 1 & .16 mg/ 100 ml) ; however, this change in total plasma calcium was significantly greater (P < .05) when compared to the change in total plasma calcium obtained in dogs receiving only an infusion of sodium chloride (group 111). Following the infusion of a sodium citrate solution into acutely TPTX dogs (group Ir), total plasma calcium concentration de- creased significantly (- -31 * .lO mg/loo ml; P < .05). The plasma phosphate concentrations in, the three groups of dogs during the hydropenic periods were not significantly different. Plasma phosphate concentration did not change following the sodium citrate infusion in group I or following the sodium chloride infusion in group III. However, plasma phosphate concentration increased significantly ( + 1.3 & .4 mg/lOO ml; P < -05) in the TPTX dogs (group II) fol- lowing the sodium citrate infusion. Associated with the infusion of sodium citrate into the blood supply of the para- thyroid gland (group I), serum immunoreactive PTH in- creased 26 A 8 &eq/ml (n = 3), which was significantly greater (P < -05) than the change in serum iPTH (-3 * 2 pl-eq/ml; n = 2) obtained in TPTX dogs receiving the same infusion of sodium citrate. Serum iPTH concen-

TABLE 1. Summary of plarma calcium and phosphate data ~__I ______- ~ -~ .~ ______.

Plasma Phosphate, mg/lOO ml

II E H E H E

Femoral Carotid

Grout I, n = 8 Intact + Na

citrate Group II, n = 5 TPTX + Na

citrate Group III, n = 7 Intact + NaCl

9.2 Ifs.1

9.7 Zk.1

9.5 Ik.2

9.5 4.03 h.3 zk.10

9.4* k.1

9.4 It.3

4.03 Zt.09

3.17* AZ.35

5.4 It.3

5.6 zk.5

6.3 zt.4

5.6 h.4

6.9* zk.4

6.3 AZ.4

Values are means & SE. H, bydropenic periods; E, experi- mental periods. * P < .05 cornDared to the hvdroDenic Deriod.

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1172 E. G. SCHNEIDER

25

*2

E H E H E H E H E I -.--_-~__________ ___. -_--

Group I (n = 8) 25 .30 .32 0.6 0.6 0.6 0.5 14 23*

*2 sf.02 zt.03 A.2 *.2 *.2 *.l *3 *3

Group II (n = 5)

30 30 .32 .33 0.6 0.8 1.3 1.1 11 13 *3 *3 zt.03 LO2 rt.4 A.3 3~5 h.6 &3 A3

Group III (n = 7)

29 zt6

26 .24 .29 1 .l 0.8 1 .O 0.8 21 24 &6 AZ.03 A.05 *.3 *.2 *.3 *.1 *3 *4

-_ .~ Values are means & SE. H, hydropenic periods; E, experi-

mental periods. * P < .025 compared- to the hydropenic period.

tration in the hydropenic periods averaged 41 A 12 pl- eq/ml for the three dogs in group I and 29 & 11 pl-eq/ml for two dogs in group II.

A summary of the clearance data is presented in Table 2. NO significant changes in glomerular filtration rate, filtra- tion fraction, or the fractional clearance of sodium or cal- cium occurred in any of the groups. The fractional clear- ance of phosphate increased 9.3 & 2.8 ml/min per 100 ml GFR (P < .025) following the sodium citrate infusion in group I. The fractional clearance of phosphate was not significantly changed in either group II or group III.

A summary of the micropuncture data is presented in Table 3. Following the infusion of sodium citrate solution (group I), fractional sodium reabsorption by the proximal tubule decreased -0.058 rt 0.18 (P < .025) which is equal to a 14% inhibition in sodium reabsorption. This change in proximal sodium reabsorption was not associ- ated with any significant change in single-nephron filtra- tion rate. In neither group II nor group III dogs were there any significant changes in fractional sodium reabsorption by the proximal tubule or in the single-nephron filtration rate.

DISCUSSION

The present study was designed to examine the effect of a selective increase in endogenous PTH secretion on proxi- mal sodium reabsorption in the dog. Several observations indicate that the infusion of sodium citrate into the para- thyroid gland increased the endogenous secretion of PTH. It has been established that PTH secretion is inversely correlated with the plasma ionized calcium concentration rather than total plasma calcium concentration (8, 15). Documentation of the perfusion of the parathyroid gland was obtained bythe injection of lissamine green dye through the infusion catheter and directly observing the perfusion of the thyroid and parathyroid glands in several of the dogs.

Following the sodium citrate infusion in dogs with in- tact parathyroid tissue, the increases in total plasma cal-

cium concentration and in phosphate excretion are con- sistent with an increase in the secretion of PTH (11).

TABLE 3. Summary of proximal tubule micropuncture data --.- - ~- _~_____~ --- ~ _- _._. -_____ ~~----_ _ -~ _____- -_____-__

Single-Nephron Dog No. No. of Fractional Reabsorption Filtration Rate, nl/min

Tubules __--- -. - H E H E

~___~~_____._ _____ ~.___ - -~. ~~___ __

Group I

I 6 2 5 3 3 4 5 5 6 6 5 7 3 8 6

.49 A-0 113 93

.45 .38 86 80

.25 .25 58 71 A-0 .32 88 85 .43 .42 102 81 .59 .45 .21 .21 56 72 .41 .34 92 71

Group II

9 5 .39 A-0 130 117 IO 4 .47 .43 80 75 II 3 .32 A0 82 80 I2 6 .41 .36 108 122 13 5 .46 .45 98 95

Group III

14 4 .39 A-0 75 78 15 5 .24 .32 89 84 16 3 .33 .21 67 79 17 2 .34 .36 89 89 18 4 .32 .33 92 97 19 4 .17 .23 59 51 20 4 .50 .47 55 61

Mean data Group I 39 .4ozt.o4 .34&03” 85&7 79sft8 Group II 23 .41+ .02 .41zk .Ol loo*9 98zk9 Group III 26 .33zt.O4 .33rt.o3 75*6 77zk6

- -_- .- --- ---- Values are means rf= SE. H, hydropenic period; E, experimental

period. * P < .05 compared to the hydropenic period.

Furthermore 75 min following the start of the sodium citrate infusion in dogs with intact parathyroid tissue there was a consistent increase in serum iPTH concentration in the three dogs in which that variable was measured. Be- cause the samples for iPTH were obtained 75 min following the start of the sodium citrate infusion, the magnitude of the change in plasma iPTH concentration probably does not accurately reflect the maximum change in serum iPTH concentration. Fischer et al. (8) have demonstrated that the maximum increase in serum iPTH concentration occurs within 4 min of a fall in plasma ionized calcium and that after 15 min the serum iPTH concentration declines somewhat from the maximum level, even though plasma ionized calcium may continue to decrease. Consequently, lowering of the plasma ionized calcium concentration per- fusing the parathyroid gland in the present study caused a significant increase in the secretion of parathyroid hormone.

Several observations suggest that following the sodium citrate infusion into dogs with intact parathyroid tissue, the small but consistent inhibition of fractional sodium re- absorption by the proximal tubule was caused by an in- crease in endogenous PTH secretion. In the absence of intact parathyroid tissue, a sodium citrate infusion did not cause a detectable inhibition of proximal sodium reab- sorption or an increase in phosphate excretion. That acute thyroparathyroidectomy prevented an increase in plasma

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PTH SECRETION AND PROXIMAL SODIUM REABSORPTION 1173

PTH concentration is supported by finding a small decrease in plasma PTH concentration following the infusion of sodium citrate in the two dogs in which that variable was measured. Additionally, the small but significant decrease in total plasma calcium concentration and the significant increase in plasma phosphate concentration are consistent with a decrease in plasma PTH concentration. The in- hibition of proximal sodium reabsorption was not associ- ated with any detectable alteration in whole-kidney or single-nephron glomerular filtration rate or in the filtration fraction for the whole kidney. The magnitude of the in- hibition of proximal sodium reabsorption is similar to that obtained in a previous study following the infusion of exogenous bovine parathyroid hormone ( 17).

It is unlikely that a small increase in total plasma calcium caused either the inhibition of proximal sodium reabsorp- tion or the increase in urinary phosphate excretion. Several investigators (3, 5, 6) have shown that large increases (6-14 mg/ 100 ml) in plasma calcium concentration cause a small inhibition in sodium reabsorption by the proximal tubule. However, the inhibition of proximal sodium re- absorption following the infusion of sodium citrate into the blood supply of the parathyroid gland was not associated with a statistically significant increase in either total plasma calcium or ionized plasma calcium concentration. Thus, it is unlikely that changes in plasma calcium concentration contributed to the observed inhibition of sodium reab- sorption by the proximal tubule. Eisenberg (7) reported that prolonged infusion of calcium solution in hypopara- thyroid patients produced increases in phosphate excretion ; other authors have reported conflicting findings. Lavender and Pullman ( 14) and Glorieux and Striver (9) found that acute increases in plasma calcium concentrations de-

REFERENCES

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crease phosphate excretion, while Cuche (4) could find no

the previous observation that infusion of exogenous PTH inhibits proximal sodium reabsorption and extends these observations by demonstrating that the endogenous re- lease of PTH can influence sodium reabsorption by the proximal tubule.

The importance of PTH in mediation of the decrease in sodium reabsorption by the proximal tubule following in- fusion of hyperoncotic albumin solution has recently been demonstrated by Knox et al. ( 13). Preferential expansion of the plasma volume failed to produce an inhibition of sodium reabsorption by the proximal tubule if the en- dogenous release of PTH was prevented. The endogenous release of parathyroid hormone was shown to mediate the inhibition of sodium reabsorption by the proximal tubule in the presence of preferential plasma volume expansion. The present study supports this conclusion and further demonstrates that the endogenous release of PTH can in- hibit sodium reabsorption by the proxi mals without preferential pl asma volume

ma1 tubule in ani- expansion.

The author gratefully acknowledges the technical assistance of Theresa Berndt, Joann Caron, Sally Gleason, and Karen Drake and the secretarial assistance of Wanda Cross.

The author also thanks Dr. C. R. Arnaud, Mayo Clinic, for per- forming the immunoassay for parathyroid hormone.

This investigation was supported in part by Public Health Service Grant HL- 16658.

E. G. Schneider is an Established Investigator of the American Heart Association.

Received for publication 29 October 1974.

11. KLEEMAN, C. R., S. G. MASSRY, AND J. W. COBURN. The clinical physiology of calcium homeostasis, parathyroid hormone, and calcitonin. Calif. Med. 114: 16-43, 1971.

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14. LAVENDER, A. R., AND T. N. PULLMAN. Changes in inorganic phosphate excretion induced by renal arterial infusion of calcium. Am. J. P/z-ysiol. 205 : 1025-1032, 1963.

15. LYNCH, R. E., E. G. SCHNEIDER, J. W. STRANDHOY, L. R. WILLIS, AND F. G. KNOX. Effect of lissamine green dye on renal sodium reabsorption in the dog. J. A/$. Physiol. 35: 169-17 1, 1973.

16. MOORE, E. W. Ionized calcium in normal serum, ultrafiltrates, and whole blood determined by ion-exchange electrodes. J. C/in. Invest 49: 3 18-334, 1970.

17. SCHNEIDER, E. G., J. W. STRANDHOY, L. R. WILLIS, AND F. G. KNOX. Relationship between proximal sodium reabsorption and excretion of calcium, magnesium and phosphate. Kidney Intern. 4: 369-376, 1973.

18. SCHNEIDER, E. G., R. E. LYNCH, L. R. WILLIS, AND F. G. KNOX. Single nephron filtration rate in the dog. Am. J. Physiol. 222: 667-673, 1972.

19. VUREK, G., AND S. PEGRAM. Fluorometric method for the determi- nation of nanogram quantities of inulin. Anal. Biochem. 16: 409- 417, 1966.

20. YOUNG, D. S. Improved method for the automatic determination of serum inorganic phosphate. J. Clin. Pat. 19: 397-399, 1966.

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