Acid-base balance in uremic rats with vascular calcification.

BACKGROUND/AIMS: Vascular calcification (VC), a major complication in humans and animals with chronic kidney disease (CKD), is influenced by changes in acid-base balance. The purpose of this study was to describe the acid-base balance in uremic rats with VC and to correlate the parameters that define acid-base equilibrium with VC.
METHODS: Twenty-two rats with CKD induced by 5/6 nephrectomy (5/6 Nx) and 10 nonuremic control rats were studied.
RESULTS: The 5/6 Nx rats showed extensive VC as evidenced by a high aortic calcium (9.2 ± 1.7 mg/g of tissue) and phosphorus (20.6 ± 4.9 mg/g of tissue) content. Uremic rats had an increased pH level (7.57 ± 0.03) as a consequence of both respiratory (PaCO2 = 28.4 ± 2.1 mm Hg) and, to a lesser degree, metabolic (base excess = 4.1 ± 1 mmol/l) derangements. A high positive correlation between both anion gap (AG) and strong ion difference (SID) with aortic calcium (AG: r = 0.604, p = 0.02; SID: r = 0.647, p = 0.01) and with aortic phosphorus (AG: r = 0.684, p = 0.007; SID: r = 0.785, p = 0.01) was detected.
CONCLUSIONS: In an experimental model of uremic rats, VC showed high positive correlation with AG and SID.

Introduction

Chronic kidney disease (CKD) is a major health problem [1,2]. Arteriosclerosis and atherosclerosis are common features in patients with CKD [3,4]. Vascular calcification (VC) significantly contributes to the high rate of cardiovascular mortality associated with CKD [5,6]. In uremic patients, VC is caused in part by a deranged mineral metabolism, including hyperphosphatemia, hypercalcemia and abnormal PTH levels [7].

Changes in acid-base balance are known to influence VC. Acidosis has been reported to decrease VC [8], whereas alkalosis is thought to increase VC [9,10]. The information on the influence of the acid-base status on VC has been obtained from two major groups of studies: (1) experiments in which the acid-base balance has been severely modified (e.g. by inducing acidosis) [8,10], and (2) epidemiological studies carried out with human patients on dialysis [11,12].

Although a great deal of knowledge about VC secondary to renal failure has been obtained from experimental models of uremic rats [13,14,15,16], to our knowledge, associations between changes in the acid-base balance and VC in uremic rats not subjected to artificial pH changes have not been reported. The present study is aimed to describe the acid-base balance in uremic rats with VC and to correlate the parameters that define acid-base equilibrium with the vascular mineral content of these rats. We hypothesize that a strong correlation between changes in the acid-base balance and VC would be found.

Methods

Animals and Surgical Procedures

Zucker ob/ob rats (n = 32) provided by Harlan Laboratories Models (Barcelona, Spain) were housed under a 12-/12-hour light/dark cycle and given ad libitum access to a standard diet (calcium = 0.6%, phosphorus = 0.6%). CKD was induced in 22 rats, and 10 rats were used as controls. For this rodent model of CKD, a 5/6 nephrectomy (5/6 Nx) was used, that is, a two-step procedure that reduces the original functional renal mass by five-sixths (5/6). In the first step, the animals were anesthetized, an 8-mm incision was made on the left mediolateral surface of the abdomen, and the left kidney was exposed. The two renal poles were tightly ligated and ablated, thus leaving 1/3 of the original renal mass. After 1 week of recovery, the animal was reanesthetized, and an 8-mm incision was made on the right mediolateral surface of the abdomen. The right kidney was exposed and unencapsulated, the renal pedicle clamped and ligated, and the kidney was removed. After the second surgery, the mineral content of the diet was changed to 0.6% calcium and 0.9% phosphorus, and the rats were treated with calcitriol 80 ng/kg i.p. (Calcijex; Abbot, Madrid, Spain) every other day (three times per week) to control secondary hyperparathyroidism. Nephrectomized rats were fed with the high-phosphate diet and treated with calcitriol for 15 days; then, they were sacrificed. Euthanasia was performed 24 h after receiving the last dose of calcitriol. All experimental protocols were reviewed and approved by the Ethics Committee for Animal Research of the University of Córdoba (Spain).

Assessment of VC

Following sacrifice, the thoracic aorta was dissected and processed to study the mineral content. Calcification was studied by measuring the aortic calcium and phosphorus content. The tissues were demineralized in 10% formic acid, and the calcium and phosphorus content was measured in the supernatant according to a method previously described [13].

Blood Chemistries

Blood for chemistry analyses was obtained from the abdominal aorta at the time of sacrifice. Blood for the measurement of the acid-base balance was collected in heparinized syringes and immediately analyzed using a Ciba-Corning 800 series blood gas analyzer (Ciba-Corning, Essex, UK). Measurements of pH, PaCO2, Na, K, Cl and ionized calcium (Ca2+) were made using selective electrodes. From these parameters, values of HCO3, concentration of total carbon dioxide (CtCO2), base excess (BE), anion gap [AG = (Na + K) – (Cl + HCO3)] and strong ion difference (SID = (Na + K) – Cl) were calculated. Afterwards, plasma was separated by centrifugation and stored at −20°C until assayed. Plasma creatinine and phosphorus were measured by spectrophotometry (BioSystems SA, Barcelona, Spain). PTH levels were quantified according to the vendor's instructions using a rat PTH(1-34) immunoradiometric assay kit (Immunotopics, San Clemente, Calif., USA). Radioimmunoassay was used to determine 1,25(OH)2D3 in plasma samples (IDS kit, Boldon, UK).

Statistics

Values are expressed as the mean ± standard error (SE). The difference between means was assessed by ANOVA. A correlation study was carried out using the Pearson test. p < 0.05 was considered significant.

Results

As shown in table 1, 5/6 Nx rats had a typical uremic blood profile, including elevated creatinine (1.36 ± 0.08 mg/dl), low Ca2+ (1.12 ± 0.06 mmol/l) and high phosphate (10.1 ± 0.5 mg/dl). These rats also showed extensive VC as evidenced by the high aortic calcium (9.2 ± 1.7 mg/g of tissue) and phosphorus (20.6 ± 4.9 mg/g of tissue) content. Both PTH (120.9 ± 19.1 pg/ml) and calcitriol (254.1 ± 49.1 pg/ml) were elevated in the 5/6 Nx rats. The control group had normal renal function, normal parameters of mineral metabolism and did not show extraskeletal calcification.

Table 1

Blood parameters of renal function and mineral metabolism, and aortic mineral content in uremic (5/6 Nx) and control rats

	5/6 Nx (n = 22)	Control (n = 10)
Plasma creatinine, mg/dl	1.36 ± 0.08	0.44 ± 0.02**
Plasma ionized calcium, mmol/l	1.12 ± 0.06	1.26 ± 0.03*
Plasma phosphate, mg/dl	10.11 ± 0.54	6.90 ± 0.44**
Plasma calcitriol, pg/ml	254.1 ± 49.1	22.4 ± 11.5*
Plasma PTH, pg/ml	120.9 ± 19.1	27.3 ± 2.7*
Aortic calcium, mg/g	9.20 ± 1.66	0.01 ± 0.01**
Aortic phosphorus, mg/g	20.60 ± 4.86	0.01 ± 0.01**

Values are means ± SE.

p < 0.05

p < 0.01 vs. 5/6 Nx.

Table 2 depicts the acid-base balance in the study animals. The elevated blood pH detected in the 5/6 Nx group (7.57 ± 0.03) revealed a tendency to alkalemia. The increased pH was a consequence of both respiratory (PaCO2 = 28.4 ± 2.1 mm Hg) and, to a lesser degree, metabolic (HCO3 = 25.1 ± 1.1 mmol/l) derangements. This alkalotic trend was also reflected in BE (4.1 ± 1 mmol/l). No major changes were found in other electrolytes. Rats from the control group showed normal acid-base parameters.

Table 2

Blood acid-base parameters and electrolytes in uremic (5/6 Nx) and control rats (see Methods for details)

	5/6 Nx (n = 22)	Control (n = 10)
pH	7.57 ± 0.03	7.36 ± 0.1**
paCO2, mm Hg	28.44 ± 2.11	44.31 ± 2.21**
HCO3−, mmol/l	25.04 ± 1.06	24.52 ± 1.32
ctCO2, mmol/l	25.95 ± 1.11	25.90 ± 1.38
BE, mmol/l	4.06 ± 1.02	−1.11 ± 1.11**
Na, mmol/l	143.08 ± 0.60	135.98 ± 1.36*
K, mmol/l	4.89 ± 0.15	4.09 ± 0.16
Cl, mmol/l	111.23 ± 1.29	106.11 ± 1.72
AG, mmol/l	10.42 ± 1.48	9.1 ± 1.3
SID, mmol/l	37.37 ± 1.97	34.01 ± 1.83

Values are means ± SE.

p < 0.05

p < 0.01 vs. 5/6 Nx.

The correlation between the aortic calcium and phosphorus content and acid-base parameters is shown in table 3. A positive correlation was found between blood pH and both aortic calcium or phosphorus concentration. PaCO2 and HCO3 showed weak negative correlations with aortic mineralization. However, the most striking fact was the high positive correlation of both AG and SID with aortic calcium (AG: r = 0.604, p = 0.02; SID: r = 0.647, p = 0.01) as well as with aortic phosphorus (AG: r = 0.684, p = 0.007; SID: r = 0.785, p = 0.01) (fig. 1).

Table 3

Correlation between aortic mineral content and blood acid-base parameters in uremic (5/6 Nx) rats (see Methods for details)

	Aortic calcium		Aortic phosphorus
	r	p	r	p
pH	0.491	0.020	0.559	0.007
paCO2	−0.542	0.011	−0.647	0.002
HCO3−	−0.407	0.060	−0.444	0.039
ctCO2	−0.424	0.049	−0.464	0.030
BE	−0.057	0.801	0.048	0.832
Na	0.261	0.368	0.475	0.086
K	−0.325	0.140	−0.461	0.031
Cl	−0.085	0.705	0.094	0.676

Fig. 1

a Correlation between aortic mineral content and AG. b Correlation between aortic mineral content and SID. Squares and the broken line represent aortic calcium (r = 0.604, p = 0.02 vs. AG: r = 0.647, p = 0.01 vs. SID), and circles and the continuous line represent aortic phosphorus (r = 0.684, p = 0.007 vs. AG: r = 0.785, p = 0.001 vs. SID).

Discussion

With the experimental protocol described here, 5/6 Nx rats were not acidotic, as classically described in uremic animals and men [11,12,17], but rather alkalotic. The reason for this alkalotic trend is unknown but may relate to the severity of extraskeletal calcification. The relationship between alkalosis and calcifications seems to be bidirectional; alkalosis promotes calcification and calcification may also increase blood pH. The rationale behind finding an increase in blood pH in animals with soft tissue calcification is that in the calcification process some mineral is diverted from the bone to the extraosseous tissue [18], and in this process there is transference of bone buffer to extracellular fluid. Nevertheless, the simultaneous presence of alkalosis and calcification reinforces the concept that acidifying the extracellular fluid is a valid therapeutic intervention to prevent and treat VC [8].

The influence of the acid-base status on VC has been studied in detail. Metabolic acidosis has been shown to protect against VC [8]. A positive correlation between blood bicarbonate levels and VC has been reported in patients on hemodialysis [19]. Alkalinization seems to potentiate vascular calcium deposition [10], but a recent study has reported that alkaline pH stabilizes primary calcioprotein particles and inhibits their transition to secondary calcioprotein particles [20]. Our results support the association between VC and alkalotic trends in uremic rats.

Although the mean values of AG in 5/6 Nx rats were within reference ranges of the control group, individual 5/6 Nx rats had elevated AG which was associated with more severe calcifications. Increased AG has been reported to be linked with a higher mortality risk in early-CKD patients [21]. Hyperphosphatemia, a major feature in uremic rats, contributes to the elevation of AG. On the other hand, the elevated AG level is likely to be influenced by an increase in unidentified/nonmeasured anions (e.g. uremic toxins). The positive correlation between AG and mineral deposition in the vasculature suggests that electrolyte equilibrium is important in the development of VC. This hypothesis is further supported by the high correlation found between SID and aortic mineral content. The mechanism that may link VC and electrolyte equilibrium is not clear, but the calcium-sensing receptor (CaSR) may be involved. Although its role in VC is not completely understood, the CaSR has been hypothesized to play a role in the development of VC [22]. The CaSR, which is expressed in the vessel wall, can sense changes in ionic strength independently of alterations in osmolality [23]. Activation of the CaSR by calcimimetics has been shown to be beneficial for preventing VC [13]. Thus, changes in the ionic balance of extracellular fluid may be influencing VC through interactions with the CaSR.

It is remarkable to note that the type of acidosis that has been reported to be beneficial for calcifications is hyperchloremic acidosis [8] in which no increase in AG is present. Thus, it would be interesting to perform further studies to determine (1) the role of unidentified anions (e.g. uremic toxins) in calcifications, and (2) the role of individual electrolytes which influence AG and SID (Na, K, and Cl) and ionic strength in VC.

In conclusion, in the present study using an experimental model of uremic rats, a weak correlation between blood pH and VC has been found, whereas VC showed a high positive correlation with AG and SID.