Nephrol Dial Transplant (2004) 19: 2061–2066
DOI: 10.1093/ndt/gfh287
Advance Access publication 8 June 2004
Original Article
Calcium ions are abnormally distributed in the skin of haemodialysis
patients with uraemic pruritus
Akishi Momose1, Shigemasa Kudo1, Motoaki Sato1, Hisao Saito1, Katunori Nagai2,
Yasuo Katabira3 and Tomihisa Funyu1
1
Department of Urology, 2Department of Pathology and 3Department of Dermatology, Oyokyo Kidney Research Institute,
Hirosaki Hospital, Hirosaki, Japan
Abstract
Background. Although a close relationship between
uraemic pruritus and serum calcium levels has been
demonstrated for some time, the degree of pruritus was
not always correlated with calcium concentrations. In
the present study, we assessed calcium ion distribution
in the skin of chronic haemodialysis patients with and
without pruritus.
Methods. We excluded patients with concomitant psoriasis or atopic dermatitis or with a previous history of
allergy, those who had an arteriovenous fistula created
prior to induction of haemodialysis, and patients with
only mild pruritus. From the enrolled 22 haemodialysis
patients, we obtained forearm skin samples during
arteriovenous fistula surgery. These patients were
divided into two groups based their grade of pruritus.
The pruritus group included patients with moderate to
severe grades of pruritus (n ¼ 11, age 64 ±13 years)
and the non-pruritus group consisted of patients
without pruritus (n ¼ 11, age 59 ±13 years). We
compared the distribution of calcium ions in the
epidermis between the two groups using the
ion-capture method (oxalate-pyroantimonate-osmium
technique). In addition, we examined and compared
the groups for thicknesses of the basal, spinous and
granular layers, as well as of the stratum corneum
using an electron microscope.
Results. The pruritus group had significantly higher
calcium ion deposition in the extracellular fluid and
cytoplasm of basal cells, and in the extracellular fluid,
nuclei and cytoplasm of spinous cells compared with
the non-pruritus group. In contrast, calcium ion depositions were similar between the two groups in the
dermis/basal layer interface, the nucleus of basal cells,
the nucleus and cytoplasm of granular cells, exterior
Correspondence and offprint requests to: Akishi Momose, MD, PhD,
Department of Urology, Oyokyo Kidney Research Institute,
Hirosaki Hospital, 90 Yamazaki, Kozawa, Hirosaki 036-8243,
Japan. Email: [email protected]
of granular cells, the granular cells/stratum corneum
interface, and in the interior and exterior of corneocytes. Although the stratum corneum was significantly
thicker in the pruritus group than in the non-pruritus
group, there were no differences in basal cell, spinal
cell or granular cell layer thicknesses.
Conclusion. In chronic haemodialysis patients with
pruritis, the calcium ion concentration in the deepest
layer of the epidermis was increased, which indicated
a disrupted calcium ion gradient in the skin. These
findings point to a role for increased skin calcium ion
concentrations in the development and/or maintenance
of uraemic pruritus. However, more extensive studies
in larger patient cohorts will be necessary to confirm
this hypothesis.
Keywords: calcium ion; end-stage renal disease;
haemodialysis; skin; uraemic pruritus
Introduction
Although a variety of treatments have been tested
for uraemic pruritus, few favourable results have
been obtained. Furthermore, the pathophysiological
mechanisms of uraemic pruritus are still unclear, and
it is not known whether it originates centrally or
peripherally [1].
Pruritus can often be relieved by parathyroidectomy [2]. However, the grade of pruritus does not
always correlate with parathyroid hormone levels,
with calcium or phosphorus concentrations in blood,
or with calcium-phosphorus products [3]. In contrast,
improved pruritus associated with low-calcium dialysis has been demonstrated [4], and pruritus with
hypercalcaemia has been reported in association
with intravenous injections of vitamin D3.
We hypothesized that there is a close relationship
between calcium and uraemic pruritus. Blood calcium
Nephrol Dial Transplant Vol. 19 No. 8 ß ERA–EDTA 2004; all rights reserved
2062
levels in healthy people are regulated by parathyroid
hormone, calcitonin and vitamin D3, and are determined by calcium balances in the kidney, bone and
gastrointestinal tract. Bone is a particularly significant
calcium storage site and provides a buffering action to
maintain constant blood calcium levels.
Haemodialysis patients experience decreased calcium
elimination and reabsorption, and decreased vitamin
D3 activation in the kidney. Blood calcium levels in
these patients are strongly affected by calcium balance
alterations caused by secondary hyperparathyroidism,
bone abnormalities such as low turnover bone disease,
ingestion of an active form of vitamin D3, and at times
intravenous vitamin D3 injection [5]. They are also
caused by ingestion of calcium carbonate through the
gastrointestinal tract and absorption from dialysate.
Normal blood calcium levels in the presence of calcium
imbalances in the kidney, bone, and gastrointestinal
tract may signal abnormal calcium balance in other
organs and tissues. In dialysis patients, calcium, phosphate and vitamin D3 levels have been linked to skin
conditions, such as subcutaneous ectopic calcification.
In the present study, we evaluated calcium ion distribution in the skin of haemodialysis patients with
and without pruritus.
Subjects and methods
Patients
The Oyokyo Kidney Research Institute, at Hirosaki Hospital,
has 475 patients with end-stage renal disease who are
undergoing maintenance haemodialysis. We conducted arteriovenous fistula surgeries on 87 chronic renal failure patients
between October 2002 and May 2003. Subjects were recruited
to the study from these 87 patients. Patients with concomitant
psoriasis or atopic dermatitis or with a previous history of
allergy, those that had arteriovenous fistula created prior to
induction of haemodialysis and patients with mild grade
pruritus were excluded.
Twenty-two patients (six females and 16 males, mean age
61±13 years) agreed to participate in the study, which was
approved by the ethical committee of the hospital. Haemodialysis was given 3–4 h at three times per week using dialysers
with a high-flux membrane. We used heparin calcium (500
U/h) as an anticoagulant. The calcium concentration in the
dialysate was 2.5 mEq/l. All haemodialysis patients had a
Kt/V of 1.2 or higher.
In patients having serum intact-PTH >200 pg/ml,
peroral administration of alfacalcidol was started at 0.25
mg/day and was incremented by 0.25 mg/day. When serum
inorganic phosphorus was >6.0 mg/dl, CaCO3 was given
as a phosphate binder. When serum calcium was
>11.0 mg/dl, alfacalcidol and CaCO3 were either decreased
or stopped.
Study protocol
At the time of arteriovenous fistula surgery, pruritus was
classified as none, mild, moderate or severe by questionnaire.
Subjects were divided into two groups based on the grade
A. Momose et al.
of pruritus. The pruritus group consisted of patients with
moderate to severe pruritus and the non-pruritus group consisted of patients without pruritus. Haemodialysis patients
with mild skin pruritus were excluded because of their
fluctuating disease, which disappeared or became more
moderate depending on time and day.
Blood was drawn early in the morning on the day of arteriovenous fistula surgery. In the two groups, we compared
serum concentrations of calcium, calcium ion, inorganic
phosphorus, parathyroid hormone (intact-PTH), 1a-25(OH)2 vitamin D, calcitonin, iron, magnesium, total bile
acid and plasma concentrations of IgE and histamine. The
serum calcium values were adjusted using the following
formula: revised serum albumin concentration ¼ (4.0 – albumin concentration) þ serum calcium concentration [6]. Serum
calcium, inorganic phosphorus, magnesium and total bile acid
were measured by an enzymatic method. Serum calcium ion
was measured by ion electrode. Serum parathyroid hormone
(intact-PTH), 1a-25-(OH)2 vitamin D and calcitonin were
assessed by RIA, serum iron by direct colorimetry, plasma
IgE by a latex turbidimetric method and plasma histamine
by HPLC.
Transepidermal water loss (TEWL) was measured prior
to surgery at 20–25 C and 25–45% relative humidity using a
Tewameter TM210 (Integral Co., Tokyo, Japan) on the forearm contra lateral to the arteriovenous fistula location.
Skin samples, of 10 5 mm, were obtained at the forearm during formation of the arteriovenous fistula. We used
an ion-capture method (oxalate-pyroantimonate-osmium
technique) to measure calcium levels in the skin, and
compared intra- and extracellular calcium ion distributions
in each skin layer between the two groups. Skin samples were
immersed in ice-cold oxalate-containing cacodylate buffer
(40 mM potassium oxalate, 140 mM KCl, pH 7.4) for 15 min
at 37 C. The primary fixative contained 40 mM potassium
oxalate, and 2.5% glutaraldehyde, adjusted to pH 7.4.
Samples were fixed overnight at 4C. Most of the dermis
was removed with a blade. The epidermis was finely minced
and then postfixed in 1% osmium tetroxide containing 2%
potassium pyroantimonate, at pH 7.4, for 2 h at 4C.
Tissue samples were then washed in alkalinized distilled
water (pH 10), and transferred to ethanol solutions for
dehydration before embedding in epoxy resin. Ultra-thin
sections (unstained) were viewed with an electron microscope.
For quantification of the calcium precipitates, a semiquantitative scale ranging from 0 (absence) to 4þ (maximum
density) was used to compare the two groups.
We also examined the perpendicular thicknesses of the
basal, spinous and granular layers, and of the stratum corneum. These were measured with an electron microscope and
were compared between the two groups.
Statistical analysis
Statistical analyses were performed using StatView version
5.0 software (Abacus Concepts, Inc., Berkeley, CA). Fisher’s
exact tests were used for categorical data for comparisons
between two groups. Results are expressed as means±SD
for normally distributed data, and as medians (total range)
for non-normally distributed data. Statistical comparisons
between the two groups were made by parametric unpaired
t-tests for normal distributions or Mann–Whitney U-tests for
non-parametric distributions. Fisher’s exact tests were applied
Cutaneous calcium ion in uraemic pruritus patients
2063
Table 1. Clinical characteristics in hemodialysis patients with and without pruritus
Characteristics
(Grading of pruritus)
Pruritus
(moderate severe)
Non-Pruritus
(absent)
Number of patients
Age
Female gender, no. (%)
Duration of hemodialysis (month)
Allergy history, no. (%)
HBV/HCV, no. (%)
Anti-allergenic/antihistamine agents use, no. (%)
Ointment use, no. (%)
Vitamin D3 use, no. (%)
Vitamin D3 (mg/day)
CaCO3, no (%)
CaCO3 (g/day)
Ca antagonist use, no. (%)
11
63.8±12.8
1 (9)
0.75 (0.25–126)
0 (0)
5 (45)
7 (64)
6 (55)
4 (36)
0 (0–0.25)
5 (45)
0 (0–4.5)
8 (73)
11
58.5±12.9
5 (45)
0.75 (0.25–194)
0 (0)
3 (27)
0 (0)
0 (0)
7 (64)
0.25 (0–0.25)
6 (55)
1.5 (0–6)
8 (73)
P-value
0.34b
0.06a
0.64c
1.00a
0.66a
0.004a
0.01a
0.39a
0.21c
1.00a
0.52c
1.00a
Data are shown mean±SD, median (total range) or number of patients (%).
Statistics: aFisher’s exact test, bthe unpaired t-test, cMann-Whitney U test.
Table 2. Biochemical data and TEWL of hemodialysis patients with and without pruritus
(Grading of pruritus)
Pruritus
(moderate severe)
Non-Pruritus
(absent)
Number of patients
Serum Ca (mg/dl)
Revised serum Ca (mg/dl)
Serum Ca2þ (mmol/l)
iP (mg/dl)
CaiP
i-PTH (pg/ml)
Vitamin D3 (pg/ml)
Calcitonin (pg/ml)
Mg (mg/dl)
Fe (mg/dl)
IgE (IU/ml)
Histamine (ng/ml)
Bile acids (mmol/l)
TEWL (g/hm2)
11
8.2±1.0
8.9±0.8
1.2±0.1
5.6±0.1
43.9 (16.3–86.5)
264 (9–878)
17.5 (5.2–31.0)
39.6 (18.7–148.8)
2.6±0.7
35 (8–84)
103 (41–1300)
0.47 (0.17–0.99)
5.0 (1.1–24.2)
12.6 (6.2–21.4)
11
8.5±1.2
9.1±1.2
1.2±0.1
6.2±1.1
57.9 (30.9–72.5)
171 (102–1729)
12.3 (4.1–17.6)
34.8 (15.0–122.4)
2.5±0.4
44 (8–100)
103 (41–897)
0.39 (0.15–7.52)
4.3 (2.0–29.2)
8.2 (4.8–10.7)
P-value
0.59a
0.69a
0.81a
0.45a
0.38b
0.92b
0.29b
0.40b
0.72a
0.69b
0.97b
0.44b
0.97b
0.03b
Values are presented as mean±SD or median (range) for variables with a skewed distribution.
Statistics: athe unpaired t-test, bthe Mann-Whitney U test. TEWL: transepidermal water loss.
Revised serum Ca ¼ (4.0 serum albumin) þ serum Ca.
for the differences in epidermal calcium ion distribution
between the two groups. P-values of P<0.05 were considered
to be statistically significant.
Results
Clinical characteristics of the patients are shown in
Table 1. There were no differences between the two
groups in age, gender, duration of haemodialysis,
number of patients with a history of allergy, number
of patients with hepatitis B or C, number of patients
taking vitamin D3, CaCO3 or using calcium channel
blockers. There were also no differences in dosages of
vitamin D3 and CaCO3. More patients in the pruritus
group used anti-histamines, allergy drugs and moisturizing ointment. There were no differences in calcium or
calcium ions in serum, or in inorganic phosphorus,
calcium phosphorus products, histamine, intact-PTH,
1a-25-(OH)2 vitamin D, calcitonin, magnesium, Fe,
IgE or total bile acid. TEWL was significantly higher
in the pruritus group (Table 2).
The pruritus group had significantly higher calcium
ion deposition in extracellular fluid and cytoplasm of
basal cells, and in extracellular fluid, nuclei and cytoplasm of spinous cells (Figure 1). There were no significant differences in calcium ion content in other
skin layers between the two groups (Table 3).
The thicknesses of the basal, spinous and granular
layers were similar in the pruritus and non-pruritus
groups (5.9±0.6 vs 5.6±0.5 mm, 13.5±2.8 vs 12.5±
4.0 mm and 6.4±1.6 vs 5.6±0.7 mm, respectively). However, the stratum corneum was significantly thicker
in the pruritus group (7.8±1.3 vs 6.1±1.2 mm, P<0.01;
Figure 2).
2064
A. Momose et al.
Fig. 1. Electron micrographs showing cytochemical localization of ionic calcium (Ca2þ) in the epidermis of haemodialysis patients. The
Ca2þ was localized by the oxalate-pyroantimonate precipitation method. Spinous cell shows fairly abundant extracellular and intracellular
precipitates (arrows) in haemodialysis patients with pruritus (A), but Ca2þ-containing precipitates are absent in stratum spinosum in
haemodialysis patients without pruritus (B). N, nucleus; M, melanin granules.
Table 3. Distribution of calcium ion in epidermis of hemodialysis patients with and without pruritus
Layers
extra/intra cell
Dermis/basal Layers interface
Basal
Intracellular
Spinous
Extracellular*
Intracellular
Granular
Extracellulary
Intracellular
Organs
Nuclei
Cytoplasma*
Nuclei*
Cytoplasma**
Nuclei
Cytoplasma
Extracellular
Granular/cornified Layers interface
Cornified
Intracellular
Extracellular
Pruritus (n ¼ 11)
Non-Pruritus (n ¼ 11)
0±
1þ
2þ
3þ
4þ
0±
1þ
2þ
3þ
4þ
9
4
2
5
1
1
0
0
0
0
7
11
11
2
2
4
6
0
0
2
2
2
1
1
0
0
0
2
3
0
2
2
6
1
1
5
1
0
0
0
3
2
0
8
8
3
8
8
5
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
11
5
6
11
0
1
1
0
0
0
6
11
11
0
5
5
0
4
5
10
0
0
0
4
0
0
0
0
0
0
5
5
0
0
0
3
0
0
0
0
1
0
0
2
0
0
11
10
7
1
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
Data are shown number of patients.
Density is from 0 to 4þ, with 0 indicating absence of precipitates and 4 indicating highest observed density of precipitates.
*: P<0.05, **: P<0.005, y: P<0.001, Fisher’s exact test.
Fig. 2. Comparison of perpendicular width of each layer in the skin
between haemodialysis patients with and without uraemic pruritus.
Discussion
A variety of studies examining the relationship between
uraemic pruritus and calcium ions have shown that
parathyroidectomy lowered parathyroid hormone,
calcium ion and inorganic phosphorus in serum, and
the calcium content of skin [7]. In addition, renal
transplantation caused increases in serum calcium and
decreases in serum inorganic phosphorus, whereas
ultraviolet B irradiation activated vitamin D3 and
reduced phosphorus levels in the skin [8]. The antipruritic mechanism of antihistaminic or anti-allergic
drugs may involve the blocking calcium ion influx into
mast cells in the skin which inhibits degranulation
[9,10]. Lipid products, which provide a substrate for
vitamin D3 formation, can create lipid bilayers in the
stratum corneum of the skin, and moisture creams
prevent water transpiration, which maintains calcium
ion gradients. Tacrolimus ointment can block the
bonds between calcium ions and calmodulin in
lymphocytes [11]. Application of a vitamin D3 analogue
(KH1060) used specifically for psoriasis to the epithelium of rats lowered calcium ion concentrations in
the upper layer of the epidermis [12].
Menon et al. [13] reported in normal skin that
calcium ion concentration increases towards the outer
Cutaneous calcium ion in uraemic pruritus patients
epidermis, forming a calcium gradient within the epidermis. The present study examining uraemic pruritus
demonstrated that the calcium ion concentration in the
deeper layer of the epidermis was significantly higher
in the pruritus group than in the non-pruritus group.
Also in the pruritis group, calcium ions were distributed
equally in all layers except for the stratum corneum,
which indicated disruption of the calcium gradient.
This may have been caused by disruption of the
permeability barrier function in the stratum corneum
due to abnormalities in phosphatides, including sphingomyelin and phosphatidylserines, or to deficiencies in
the permselectivity of the basement membrane toward
calcium ions. In agreement with our study, Menon et al.
[13] showed that calcium ions were present in all layers
of the skin in essential fatty acid deficient or topical
lovastatin-treated mice, implicating a disruption of the
ion gradient. In chronic forms of skin barrier defects,
which differ from acute defects, the calcium ion concentration in the inner layer of the skin increases so that
water flow towards the epidermis [14].
We found a higher TEWL in haemodialysis patients
with pruritus compared with patients without pruritus.
However, these values were all within the normal range.
TEWL was lower in senile xeroderma than in healthy
elderly subjects [15], suggesting an abnormally high
skin barrier function caused by aberrant phosphatide
in the stratum corneum. However, and in contrast to
our findings, Ostlere et al. [16] reported no difference in
TEWL values between uraemic pruritus patients and
non-pruritus patients. Thus, disruption of the calcium
concentration gradient in the skin of haemodialysis
patients with pruritus may be caused by an abnormal
permeability barrier function of the corneocyte along
with an abnormal calcium ion selectivity function of
the basal membrane cell layer.
While examining normal skin, Hennings et al. [17]
reported that low levels of extracellular calcium ions
may accelerate differentiation of keratinocytes and that
high extracellular calcium ion concentrations may play
an important role in the proliferation of keratinocytes.
Although psoriasis patients do not normally experience
pruritus, the concentration of calcium ions in the
deeper layer of the epidermis of these patients is lower
than that in healthy subjects, and increased proliferation and abnormal differentiation of keratinocytes
have been observed [13]. Although we did not carry
out comparative examinations with healthy subjects,
the concentration of calcium ions in the deeper layer of
the epidermis was significantly higher in haemodialysis
patients with uraemic pruritus than in non-pruritus
haemodialysis patients. In addition, only the stratum
corneum of haemodialysis patients suffering uraemic
pruritus was thicker than in non-pruritis patients. Thus,
in patients with uraemic pruritus, as opposed to psoriasis patients, differentiation may be accelerated by
higher concentrations of calcium ions in the deeper
layer of the epidermis. It is possible that patients with
pruritus had a significantly thicker stratum corneum
because of elevated ion concentrations, and that
scratching may have removed thickened corneocytes.
2065
However, it is also possible that the stratum corneum of
haemodialysis patients with pruritus is thickened by
chronic scratching.
In previous work, the skin surface of haemodialysis
patients with pruritus appeared normal when the
disease was not associated with scratches, scars or
secondary skin disorders [18]. In contrast to atopic
dermatitis associated with pruritus, the normal appearance of the skin surface may be due to an
anomaly in the inner layer of the skin and not on the
surface of the skin.
It is not clear whether the observed increases in calcium ions in the deeper layer of the epidermis are a
cause of uraemic pruritus or only a related phenomenon. Johansson et al. [19] reported that neuronspecific enolase immunoreactive nerve fibres were
distributed beyond the basement membrane to
the epidermis in 12 dialysis patients but these were
not observed in 15 controls. In contrast, Fantini et al.
[20] revealed that neuron-specific enolase immunoreactive nerve fibres within the epidermis were present in
the great majority of healthy subjects, but in only three
out of 24 uraemic patients. In another study, haemodialysis patients with pruritis had increased calcium
ions in the extracellular fluid around the receptors of
sensory nerve endings within the spinous layer, the
basement membrane and the dermis because calcium
ions in the extracellular fluid within the dermis were at
high levels [21].
Calcium ion concentrations in extracellular fluid
have been shown to influence sensory nerve terminals
either directly by voltage-sensitive ion channel gates
according to the surface potential theory [22], or
through indirect mechanisms. The threshold for sensing pruritus stimuli may be lowered by changes in the
impulse activity of unmyelinated C-nerve fibres, which
transmit and integrate pruritus, pain and cold sensations [23]. This mechanism appears to be in agreement
with the currently accepted pattern theory [24].
A number of potential uraemic pruritogens have
been reported by several research groups [25]. An
in vitro study showed that mast cells, macrophages,
lymphocytes and keratinocytes secrete cytokines,
including interleukin 2, and proteases, such as histamine, tryptase and chymase when calcium ion concentrations in the extracellular fluid were high [26].
Therefore, high calcium ions in the extracellular fluid of
the inner layer of the epidermis may stimulate excessive
production of cytokines that induce the release of
pruritogenic substances [27].
In conclusion, we found an association between
increased calcium ion concentrations in the inner layer
of the epithelium and the presence of pruritus in
haemodialysis patients. Although the increase in calcium ions in the deeper layer of the epidermis is
probably not a direct cause of uraemic pruritus, we
believe that changes in the microenvironment around
the sensory nerve receptors plays a role in uraemic
pruritus development. Further investigations using
larger sample sizes will be necessary to evaluate these
possibilities.
2066
Acknowledgements. The authors would like to thank Naoto
Oyama, Masako Isono and Shunsuke Narita for their technical
assistance. We are grateful to Prof. Katsumi Hanada for helpful
discussions and comments on the manuscript.
Conflict of interest statement. None declared.
A. Momose et al.
13.
14.
15.
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Received for publication: 16.10.03
Accepted in revised form: 6.2.04