Hyperparathyroidism, platelet intracellular free calcium and hypertension in chronic renal failure

Kidney International, Vol. 43 (1993), pp. 700—705

Hyperparathyroidism, platelet intracellular free calcium and hypertension in chronic renal failure ANTHONY E.G. RAINE, LINDSEY BEDFORD, ALEC W.M. SIMPSON, CHRISTOPHER C. ASHLEY, RICHARD BROWN, J. STUART WOODHEAD, and JOHN G.G. LEDINGHAM Nuffield Department of Clinical Medicine, John Radcl(tfe Hospital, and University Laboratory of Physiology, Oxford, England, and Department of Medical Biochemistry, University of Wales College of Medicine, CardiJJ Wales, United Kingdom

hypothesis, increases in total calcium content in the heart and

Hyperparathyroidism, platelet intracellular free calcium and hyperten-

sion in chronic renal failure. To investigate possible relationships elsewhere have been documented in both clinical [8] and between hyperparathyroidism, alterations in intracellular free calcium concentration ([Ca2j1) and hypertension in chronic renal failure, serum concentrations of intact parathyroid hormone (PTH) were measured by two-site immunometric assay, and platelet ([Ca2]1) was assessed using the fluorescent indicator fura-2. Thirty-six patients with chronic renal failure were studied, 10 with normal serum PTH concentrations (mean 8.0 0.6 pmollliter), 17 with elevated serum PTH (35.0 7.2 pmollliter) and 9 patients with elevated PTH (36.2 5.9 pmol/liter) who

were receiving nifedipine. Platelet [Ca2] was increased in patients with elevated PTH, compared with those in whom PTH was normal 16 vs. 83 7 nmol/liter, P < 0.01). A linear relation was (138 observed between serum VFH and platelet [Ca21 in these patients (r = 9 0.818, P < 0.001). In contrast, platelet [Ca2] was not elevated (84 nmol/liter) in the patients with elevated PTH who were receiving nifedipine. A linear relation was also present between both serum PTH (r = 0.616, P < 0.001) and platelet [Ca2] (r = 0.576, P < 0.005) and

mean blood pressure. Nine patients with hyperparathyroidism were

restudied after treatment with the vitamin D analogue alfacalcidol. This resulted in significant decreases in serum PTH (P < 0.01), platelet [Ca2] (P < 0,02), and mean blood pressure (P < 0.05). These studies

indicate that 1Ca2] may be increased early in renal failure, and that this increase occurs in association with both hyperparathyroidism and hypertension. Furthermore, treatment of hyperparathyroidism with

experimental [9] renal failure, and these may be prevented by parathyroidectomy [9]. Cytosolic free calcium, which plays a central role in intracellular signaling, represents only a very small fraction of total

cellular calcium. Increases in cytosolic calcium have been reported in experimental models of uremia [10] and in platelets of patients with chronic renal failure [11], but the relationship of this abnormality to the secondary hyperparathyroidism of uremia remains unknown. It is also not known whether alterations

in cytosolic calcium in uremic patients are associated with increases in blood pressure, as is the case in essential hypertension [12—141.

In the present study we have therefore investigated the possible relationship of intact serum PTH to platelet intracellular free calcium ([Ca2I1) and to blood pressure in patients with chronic renal failure. In addition, the effects of the reversal of hyperparathyroidism by alfacalcidol treatment on platelet intracellular free calcium and on blood pressure have been studied.

alfacalcidol may result in reductions in both [Ca2]1 and blood pressure.

The lack of elevation in [Ca2]1 in nifedipine-treated patients with hyperparathyroidism suggests that, in uremia, increases in cytosolic calcium induced by PTH or other factors may be mediated in part by dihydropyridine-sensitive mechanisms.

Methods

Patients

Thirty-six Caucasian patients were included in the study, 23 males, 13 females, aged 18 to 77 years, mean 52 years. All had chronic renal failure but none had end-stage disease requiring maintenance dialysis. Details of patients are given in Table 1, Disordered calcium metabolism is very common in renal including the etiology of renal failure and drug treatment; this disease, and abnormalities of calcium, phosphate, vitamin D was witheld on the day of study. The patients were divided into and parathyroid hormone may all occur. In the extreme case in three groups with: (a) serum PTH concentration less than 10 end-stage renal failure, this may lead to severe and widespread pmollliter (range 4.7 to 9.9 pM; N = 10), (b) serum PTH extraosseous calcification [1]. However, secondary hyperpara- concentration greater than 10 pmol/liter (range 11.0 to 106.8 pM; thyroidism often develops early in the course of chronic renal N = 17), and (c) patients with serum PTH concentrations impairment [2, 3], and it has been suggested that parathyroid greater than 10 pmollliter (range 10.8 to 57.5 pM) who were hormone (PTH) may itself act as a uremic toxin [41 involved in receiving nifedipine therapy (N = 9). The groups were matched the neuropathy [5], cardiac dysfunction [6, 7], and other disor- for age and sex distribution, and degree of renal impairment. At ders which may occur in renal failure. In support of this the beginning of the study no patient was receiving treatment with alfacalcidol or other vitamin D analogue, and none had had parathyroidectomy. Patients with evidence of autonomous hyperparathyroidism were excluded. Received for publication March 6, 1992 and in revised form October 1, 1992 Blood samples were taken free-flowing, with the subject in Accepted for publication October 1, 1992 the seated position, between 9:00 and 10:30 a.m. For measurement of serum PTH, 10 ml whole blood was allowed to stand for © 1993 by the International Society of Nephrology 700

701

Raine et a!: [Ca2]1 in CRF

Table 1. Patient data

at 37°C. After the incubation the PRP was centrifuged at 350 x

g for 20 minutes and the platelet pellet resuspended in a

Study group Normal PTH

Patient characteristic Mean age (range) Sex M/F Diagnosis no. of patients Glomerulonephritis Pyelonephritis Polycystic kidney disease Diabetes mellitus Hypertension Other Therapy no. of patients Beta-blocker Diuretic ACE inhibitor Vasodilator Phosphate binder Insulin Other Mean PTH pmol/liter Mean creatinine .unol/liter

(N =

10)

Elevated PTH

(N =

17)

Elevated PTH + nifedipine

(N = 9)

55 (44—69)

52 (18—66)

53 (27—70)

7/3

10/7

6/3

2 5 3 3 2 2

1 1

physiological saline (containing; (mM) 140 m sodium chloride,

5 m potassium chloride, 1 m magnesium sulphate, 10 mM glucose, 10 mivi HEPES (pH 7.4), 100 LM aspirin, 20 sg/ml apyrase and 0.05 U/ml hirudin), to which 1 m calcium chloride (final) was subsequently added. The fluorescence from 1 ml aliquots, excited at 340 nm, was monitored at 500 nm in a Shimadzu spectrofluorimeter and was calibrated as follows; the

1

2 3 1 1

2

4 6

—

4 6 10 3 7

6 1

6

chloride to quench the signal giving FMfl. Fmax was taken as FdIS — Fik, Fmin as [(Fmax — FMfl) 0.37 + FMfl] and Fr as F0 — Fik.

4 7 0 5

10

1

8.0 586

2 3 2

8

2

signal from each sample (F0) was recorded and the contribution to the signal from extracellular dye (Fik) determined by adding 200 M manganese chloride. A total of 400 M DTPA was then added to chelate the manganese; 50 sM digitonin was added to lyse the cells giving Fdtg, followed by 2 m manganese

5 3 1

0.6

35.0

7.2

36.2

121

571

46

653

5.9 67

A value of 224 n was used as the Kd for fura-2 and Ca2 [17]. Using these values [Ca2]1 was calculated as described for quin2 [18] with the equation [Ca2], = Kd [(Fr — Fmin)/(Fmax — Fr)]. The emission spectra of the fura-2 loaded cells, examined for each patient's samples, showed no indication of Ca2insensitive forms of the dye as reported for polymorphonuclear leukocytes [191. All determinations of platelet intracellular free calcium were completed within four hours of obtaining blood samples.

30 minutes and then centrifuged and serum stored at —70°C until assay. For preparation of platelet-rich plasma and subsequent measurement of platelet intracellular free calcium, 5 to 10 ml blood was anticoagulated with 1/6 volume of acid-citrate dextrose containing 65 m citric acid, 85 m sodium citrate and 110 mM dextrose. All measurements of serum PTH and of platelet cytosolic calcium were carried out without knowledge of the clinical status of the patient. Ten ml heparinized blood was also taken for measurement of total and ionized plasma calcium, plasma phosphate and alkaline phosphatase. Blood pressure was recorded twice with a five-minute interval using a 'Copal' automated digital sphygmomanometer with the subject seated, after 10 minutes rest. Diastolic pressure was recorded as Korotkoff phase 5. Mean blood pressure was calculated as

Plasma ionised calcium was determined with an Ion 83

lonmeter electrode (Radiometer). Plasma total calcium, phosphate, creatinine and alkaline phosphatase were measured with an RA1000 automated analyzer. Statistics All results are expressed as mean SEM. Statistical comparison was carried out using Student's t-test for paired or unpaired normally distributed data, the Wilcoxon rank test for non-parametric analysis, and regression analysis where indicated. Results

Serum parathyroid hormone concentrations Serum PTH levels in the 36 patients ranged from 4.7 to 104.8 follow-up studies were carried out during treatment with alfacalcidol (0.25 to 1.0 g daily, mean 0.44 gIday) for a period of pmol/liter. Significant hyperparathyroidism was present in the majority of patients, including those with relatively mild renal 2 to 11 months (mean 6.2 months). failure (Fig. 1). Plasma total and free calcium, total phosphate and alkaline phosphatase are shown in Table 2 for the three Analytical procedures groups of patients. Patients with hyperparathyroidism (PTH Assay of serum PTH. Intact circulating PTH was measured greater than 10 pmollliter), whether treated with nifedipine or by a recently developed direct immunoassay described in detail elsewhere [15]. The principle of the method involves formation not, tended to have lower total and free plasma calcium and

(diastolic pressure + 1/3 pulse pressure). In nine patients

of an immune complex of labeled anti-amino terminal (1-34) phosphate and higher alkaline phosphatase than patients with PTH antibody, intact human PTH and solid phase anti-mid essentially normal parathyroid function, although these differregion (44-68) PTH antibody, with use of a chemiluminescent ences did not achieve statistical significance. aryl acridinium ester as tracer. One hundred microliter samples Serum PTH and platelet intracellular free calcium of serum were used. Assay detection limit was 0.3 pmollliter of Platelet [Ca2i was significantly increased in the 17 patients 1-84 PTH, and normal range 0.8 to 8.5 pmol/liter, determined in with increased PTH levels; 138 16 nmollliter (range 75 to 344 82 subjects [15]. 7 nmol/liter (range 48 to 118 Measurement of platelet intracellular free calcium. Platelet nmol/liter) compared to 83 rich plasma (PRP) was prepared from 5 to 10 ml samples of nmol/liter; P < 0.01) in patients with normal PTH. However, whole blood as previously described [16]. The PRP was then platelet [Ca2I1 in the nine nifedipine-treated patients was not incubated with 1 jsM fura-2 acetoxy methyl ester (fura-2/AM: elevated (84 9 nmol/liter) despite similar increases in PTH in Molecular Probes Inc., Eugene, Oregon, USA) for 45 minutes both these patients and the 17 hyperparathyroid patients (36.2

702

Raine el a!: [Ca2] in CRF Table 2. Data for the three groups of patients

.

Study group

100

Elevated Normal PTH

(N = 10) 80

Serum PTH pmol/

liter Plasma total calcium mmol/liter Plasma free calcium mmol/liter Plasma phosphate mmol/liter Plasma alkaline phosphatase

60 -

I 0 I—

E

40

Elevated PTH

(N =

17)

PTH + nifedipine (N 9)

8.0

0.6

35.0

7.2

36.2

5.9

2.34

0.06

2.27

0.05

2.28

0.05

0.96

0.07

0.91

0.04

0.90

0.06

1.59

0.20

1.43

0.08

1.54

0.16

222

27

309

49

308

38

83

7

138

16

84

9

150

4 2 3

163

7 4 4

156

6 5 6

N 100—300 i.u./liter

Platelet intracellular free calcium nmol/liter Blood pressure mm

C')

Hg

20

200

Systolic Diastolic Mean

.

.

400

600

86 107

92 116

83 107

800

Effect of vitamin D therapy Nine hyperparathyroid patients were re-studied during treatFig. 1. Serum PTH concentrations in relation to serum creatinine ment with alfacalcidol. Alfacalcidol therapy was associated concentrations in the 36 patients studied. Hatched area indicates normal range for PTH, measured by two-site immunometric assay [lii. with a fall in serum PTH concentrations from 37 11 to 14 5 pmollliter (P < 0.01) and in platelet [Ca2]1 from 157 28 to 78 8 nmol/liter (P < 0.02). The changes in individual patients are illustrated in Figure 5, which shows that in all cases, a fall in PTH produced by vitamin D analogue therapy was associated and 35.0 pmol/liter, respectively). For all patients, excluding with a fall in platelet [Ca2]1, and conversely, an increase in those receiving nifedipine therapy, there was a linear relation PTH with a rise in platelet [Ca2]1. Mean blood pressure also fell significantly with alfacalcidol therapy (122 3 to 112 3 between serum PTH concentrations and platelet [Ca2j1 (r mm Hg; P < 0.05). The absolute change in mean blood pressure 0.818; P < 0.001) as illustrated in Figure 2. during the treatment period was linearly related to both change in serum PTH (r = 0.653; P < 0.05) and change in platelet Serum PTH, platelet intracellular free calcium and blood [Ca2]1 (r = 0.660; P < 0.05). pressure Discussion Systolic, diastolic and mean blood pressure were higher in the 17 hyperparathyroid patients than in the 10 patients with The present study shows that intact serum parathyroid hornormal PTH (Table 2). When these 27 patients were subdivided mone levels and cytosolic free calcium may both become according to whether platelet intracellular free calcium was elevated early in the course of chronic renal failure, and both are greater or less than 100 nmol/liter, mean blood pressure was directly correlated. Although the causes of hyperparathyroidism higher in those with increased platelet [Ca2] (119 5 mm Hg, in chronic renal failure are complex [20], those contributing to this N = 14) than in patients with low platelet [Ca2]1 (106 2 mm abnormality in early renal failure include hypocalcemia secondary Hg, N = 13; P < 0.05). Mean blood pressure was linearly to impaired renal hydroxylation of cholecalciferol [211 and renal related to both serum PTH (r = 0.616, P < 0.001), and to phosphate retention [22]. A direct association was observed platelet [Ca2] (r = 0.576, P < 0.005), as shown in Figures 3 between serum PTH and platelet [Ca2]1 but it is not known and 4. whether a causal relationship exists between these two variTo assess whether the low platelet [Ca2] in nifedipine- ables, nor by what mechanism increased PTH might result in Serum creatinine, fimol/ilter

treated patients was related to blood pressure reduction or elevated platelet [Ca2]1. In cultured renal proximal tubule specifically to calcium entry blocker therapy, these patients cells, PTH caused an immediate increase in [Ca2} through a were compared to four hyperparathyroid patients receiving cyclic AMP-independent mechanism [23] involving stimulation hydralazine. Despite similar mean blood pressure (112

6 mm

Hg) and serum PTH (38.0 13.3 pmollliter) in the latter, platelet [Ca2] was elevated at 140 30 nmollliter.

of production of inositol triphosphate and diacyl glycerol [24], whereas in an osteoblast-like cell line, acute elevation of [Ca2j1 in response to PTH occurred through activation of both cyclic

Raine et al: [Ca2]1 in CRF

703

350

300

0 250 E

0

200

c,) (I) ci)

150 100

cci

a-

.s

50

20

60

40

80

Fig. 2. Relation between serum PTH 120 concentration and platelet intracellular free

100

calcium concentration in 27 patients (y =

Serum PTH, pmol/Iiter

E

150

150

140

140

130

E a-

120

C

110

cci

x +71.6). r = 0.818; P < 0.001.

1.8,

130

..

E E a-

120

C

110

co

ci)

cci

100 :,.

ci)

100

90

20

40

60

80

100

120

Serum PTH, pmol/liter Fig. 3. Relation between serum PTH concentration and mean blood

pressure in 27 patients (y = 0.35, x +103.9). r = 0.616; P < 0.001.

AMP-dependent and independent channels [25, 26]. It is unlikely that increased cAMP mediates the elevation in platelet

90 'I

50

100 150 200 250 300 350

Platelet resting [Ca21, nmol/liter Fig. 4. Relation between platelet [Ca2 *]. and mean blood pressure in 27 patients (y = 0.15, x +95.3). r = 0.576; P < 0.005.

D may itself increase [Ca2]1 [301 and enhance cellular calcium

[Ca2] which we observed; PTH has no apparent effect on uptake [311. Comparison of the levels of serum PTH and [Ca2] platelet cAMP in vitro [27], and in addition, cAMP in the in patients receiving or not receiving nifedipine (Table 2) platelet is an inhibitory second messenger, associated with suggests that chronic nifedipine therapy may, in contrast to other vasodilators, also prevent elevation of [Ca2] in uremic reductions in [Ca211 [281. In patients treated with alfacalcidol in the present study, patients, even when hyperparathyroidism is present. The mechincreases in both serum PTH and platelet [Ca2] were restored anism of this effect also remains unknown. Nifedipine has little towards normal. The reduction in serum PTH levels in response or no acute effect on resting or ADP-stimulated [Ca2], in to alfacalcidol is presumed due to both an increase in plasma human platelets [32], although in osteoblastic cell lines, imme-

calcium, through increased gut absorption of calcium, and diate increases in cytosolic calcium induced by PTH were direct suppression of parathyroid hormone secretion by vitamin D [291. Although the possibility of a direct effect of alfacalcidol therapy on platelet [Ca2]1 cannot be excluded, it seems more likely that the fall in platelet [Ca2] was related in some way to the concomitant reduction in serum PTH levels, since vitamin

inhibited by calcium channel blockers [26, 331. The implication

is that increases in cytosolic calcium in uremia, whether induced by PTH or by other factors, may be mediated in part by dihydropyridine-sensitive mechanisms. Relationships between both serum PTH and platelet cytosolic

704

Raine et al: [Ca2'71 in CRF

350 300 250

0

200

C

150 a)

a) Ce

100

50 Fig. 5. Effects of 2 to 11 months' treatment with alfacalcidol in 9 patients on serum PTH

40

60

80

100

12

Serum PTH pmol/liter

concentration and platelet i cellular free calcium concentration. Symbois are: (•) values before commencement of alfacalcidol; (0) corresponding values after treatment.

calcium and blood pressure in patients with renal failure were as experimental studies have previously suggested [391. The observed in the present study. In addition, alfacalcidol therapy relationships observed between PTH, intracellular calcium and was associated with a reduction in blood pressure, which was blood pressure were such that an increase in PTH from 10 to 50 related to the decreases in both serum PTH and in platelet pmol/liter was associated with a doubling of platelet [Ca2]1 [Ca2], in response to this therapy. Previous studies have from 100 to 200 nmollliter, and a 15 mm Hg increase in mean demonstrated a relationship in essential hypertension between blood pressure. In essential hypertension, the platelet [Ca2]1blood pressure and platelet [Ca2]1 [12, 13], though not lympho- blood pressure relationship is steeper; an increase in [Ca2]1 cyte [Ca2]1 [13, 34]. The mechanism of this relationship from 100 to 200 nmollliter relates to a mean blood pressure remains unknown. However, recent studies in patients with increase of 60 mm Hg [12, 13]. essential hypertension have shown relationships between intact In summary, the present study shows that cytosolic free PTH, platelet [Ca2] and blood pressure similar to those calcium, assessed in platelets, may be increased relatively early reported here [14]. Further studies in normotensive subjects in the course of chronic renal failure, and this increase is have confirmed a correlation between both intact PTH within associated with elevation of serum PTH. The associations also the normal range and platelet [Ca2] and blood pressure [35]. observed between both PTH and platelet cytosolic calcium and PTH was itself related to platelet [Ca2]1 in hypertensive blood pressure, together with the reductions in all these three

patients [14] but not normotensive subjects [35]. In these variables during the course of vitamin D therapy, suggest studies, neither body mass index nor serum calcidiol nor mutual interrelationships which merit further investigation. calcitriol were related to blood pressure. Thus, both the findings of Brickman et al [14, 35] and those of the present study suggest Acknowledgments that one factor acting to elevate platelet [Ca2]1 in hypertension AEGR was the recipient of a British Heart Foundation Senior and uremia may be serum PTH. However, it is likely that other Research Fellowship. LB and AWMS were supported by the Welicome factors such as changes in cell calcium transport mechanisms or Trust. This study was presented in part at the 21st Annual Meeting of the American Society of Nephrology, membrane permeability in uremia may also contribute.

An alternative possibility is that increased platelet [Ca2]1 occurs secondary to the development of hypertension. If this Reprint requests to Dr. A.E.G. Raine, Department of Nephrology, were the case, however, the fall in platelet [Ca2] with alfacal- St. Bartholomew's Hospital, West Smithfield, London, England ECJA cidol therapy (Fig. 5) would imply that alfacalcidol exerted a 7BE, United Kingdom. direct hypotensive effect. This appears unlikely, since serum References calcitriol is elevated in essential hypertension [14, 36], and administration of calcitriol for three days enhanced vascular I. ALFREY AC, SOLOMON CC, CouciLLo J, MILLER NL: Exsmooth muscle contractility in spontaneously hypertensive rats traosseous calcification. Evidence for abnormal pyrophosphate metabolism in uraemia. J Cliii Invest 57:692—699, 1976 and in their normotensive controls [37]. The importance of both plasma volume status and plasma 2. ARNAUD CD: Hyperparathyroidism and renal failure. Kidney mt

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