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, 2 Department of Pathology and 3 Department of Dermatology, Oyokyo Kidney Research Institute, Hirosaki Hospital, Hirosaki, Japan

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: momose-a{at}oyokyo.jp



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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 = 11, age 64 ±13 years) and the non-pruritus group consisted of patients without pruritus (n = 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 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
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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 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
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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 µg/day and was incremented by 0.25 µg/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 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), 1{alpha}-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 = (4.0 – albumin concentration) + 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), 1{alpha}-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 x 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 4°C. 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 4°C. 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 4+ (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 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
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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, 1{alpha}-25-(OH)2 vitamin D, calcitonin, magnesium, Fe, IgE or total bile acid. TEWL was significantly higher in the pruritus group (Table 2).


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Table 1. Clinical characteristics in hemodialysis patients with and without pruritus

 

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Table 2. Biochemical data and TEWL of hemodialysis patients with and without pruritus

 
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).



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Fig. 1. Electron micrographs showing cytochemical localization of ionic calcium (Ca2+) in the epidermis of haemodialysis patients. The Ca2+ 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 Ca2+-containing precipitates are absent in stratum spinosum in haemodialysis patients without pruritus (B). N, nucleus; M, melanin granules.

 

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Table 3. Distribution of calcium ion in epidermis of hemodialysis patients with and without pruritus

 
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 µm, 13.5±2.8 vs 12.5±4.0 µm and 6.4±1.6 vs 5.6±0.7 µm, respectively). However, the stratum corneum was significantly thicker in the pruritus group (7.8±1.3 vs 6.1±1.2 µm, P<0.01; Figure 2).



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Fig. 2. Comparison of perpendicular width of each layer in the skin between haemodialysis patients with and without uraemic pruritus.

 


   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
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 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. 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 neuron-specific 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.



   Acknowledgments
 
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.



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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 16.10.03
Accepted in revised form: 6. 2.04





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