Intracellular calcium increases in vascular smooth muscle cells with progression of chronic kidney disease in a rat model

Abstract

Background: Vascular smooth muscle cells (VSMCs) exhibit phenotypic plasticity, promoting vascular calcification and increasing cardiovascular risk. Changes in VSMC intracellular calcium ([Ca 2+ ] i ) are a major determinant of plasticity, but little is known about changes in [Ca 2+ ] i in chronic kidney disease (CKD). We have previously demonstrated such plasticity in aortas from our rat model of CKD and therefore sought to examine changes in [Ca 2+ ] i during CKD progression.

Materials and methods: We examined freshly isolated VSMCs from aortas of normal rats, Cy/+ rats (CKD) with early and advanced CKD, and advanced CKD rats treated without and with 3% calcium gluconate (CKD + Ca 2+ ) to lower parathyroid hormone (PTH) levels. [Ca 2+ ] i was measured with fura-2.

Results: Cy/+ rats developed progressive CKD, as assessed by plasma levels of blood urea nitrogen, calcium, phosphorus, parathyroid hormone and fibroblast growth factor 23. VSMCs isolated from rats with CKD demonstrated biphasic alterations in resting [Ca 2+ ] i : VSMCs from rats with early CKD exhibited reduced resting [Ca 2+ ] i , while VSMCs from rats with advanced CKD exhibited elevated resting [Ca 2+ ] i . Caffeine-induced sarcoplasmic reticulum (SR) Ca 2+ store release was modestly increased in early CKD and was more drastically increased in advanced CKD. The advanced CKD elevation in SR Ca 2+ store release was associated with a significant increase in the activity of the sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA); however, SERCA2a protein expression was decreased in advanced CKD. Following SR Ca 2+ store release, recovery of [Ca 2+ ] i in the presence of caffeine and extracellular Ca 2+ was attenuated in VSMCs from rats with advanced CKD. This impairment, together with reductions in expression of the Na + /Ca 2+ exchanger, suggest a reduction in Ca 2+ extrusion capability. Finally, store-operated Ca 2+ entry (SOCE) was assessed following SR Ca 2+ store depletion. Ca 2+ entry during recovery from caffeine-induced SR Ca 2+ store release was elevated in advanced CKD, suggesting a role for exacerbated SOCE with progressing CKD.

Conclusions: With progressive CKD in the Cy/+ rat there is increased resting [Ca 2+ ] i in VSMCs due, in part, to increased SOCE and impaired calcium extrusion from the cell. Such changes may predispose VSMCs to phenotypic changes that are a prerequisite to calcification.

Keywords: calcium signaling; cell phenotype; chronic kidney disease; rat model; vascular smooth muscle cells.

FIGURE 1

Serum mineral and bone disorder parameters at 10- and 35-week-old animals. The 10-week CKD animals have estimated kidney function of 75% of normal, whereas by 35 weeks of age the kidney function is 15% of normal. (A) At 10 and 35 weeks there was no difference in serum calcium among the groups. (B) Serum phosphorus was significantly greater in 35-week CKD and CKD + Ca animals compared with normal animals but not different at 10 weeks of age. (C) At 35-weeks, CKD animals demonstrated secondary hyperparathyroidism with significantly greater PTH compared with normal animals and treatment with calcium suppressed PTH. At 10 weeks of age there was no evidence of secondary hyperparathyroidism. For all panels: NL = normal (n = 6); CKD = chronic kidney disease (n = 6); CKD + Ca = chronic kidney disease + Ca²⁺ treatment (n = 8). *P < 0.05 versus 35-week normal, **P < 0.05 versus 35-week CKD.

FIGURE 2

Intracellular Ca²⁺ handling is altered with CKD. VSMCs were freshly isolated, loaded with fura-2 and imaged on an epifluorescent inverted microscope. (A) Representative tracing outlining experimental protocol described in detail in the methods section. Cells were initially perfused with physiologic saline solution (PSS) for the first minute and the average [Ca²⁺]_i during this period was defined as the baseline [Ca²⁺]_i, denoted as ‘i’. A total of 80 mM K⁺ was added, followed by washout with PSS, with the amplitude as a measure of VGCCs, denoted as ‘ii’. Cells were then transitioned into either PSS or a Ca²⁺-free solution for 1 min and then the SR Ca²⁺ store was emptied with 5 mM caffeine both in the presence and absence of extracellular Ca²⁺. SR Ca²⁺ store release was calculated as the peak caffeine [Ca²⁺]_i level in the absence of extracellular Ca²⁺ subtracted from the [Ca²⁺]_i level preceding caffeine perfusion, denoted as ‘iii’.The activity of SERCA was assessed by the magnitude of the undershoot (below baseline), denoted as ‘iv’. The recovery ‘shoulder’ was measured to determine a role for store-operated Ca²⁺ entry (SOCE) following caffeine-induced SR Ca²⁺ store release in the presence and absence of extracellular Ca²⁺ at 8 min 15 s and is expressed as a percentage of SR Ca²⁺ store release, denoted as ‘v’. (B) Resting (baseline) [Ca²⁺]_i is decreased in early (10 weeks) CKD and elevated in late (35 weeks) CKD. This was not significantly affected by the administration of calcium to lower PTH (CKD + Ca). (C) SR Ca²⁺ store release is elevated in early (10 weeks) and late (35 weeks) CKD and is partially reversed with Ca²⁺ treatment. (D) SERCA function, as assessed by recovery of [Ca²⁺]_i, was unchanged in early (10 weeks) CKD, and was increased in late (35 weeks) CKD. (E) The recovery ‘shoulder’ following SR Ca²⁺ store release in the absence of extracellular Ca²⁺ was not different between any group (0Ca; white bars). However, in the presence of extracellular Ca²⁺ (2Ca; black bars), the recovery ‘shoulder’ was increased with CKD. For all panels: NL = normal (n = 6); CKD = chronic kidney disease (n = 6); CKD + Ca = chronic kidney disease + Ca²⁺ treatment (n = 8). *P < 0.05 10-week CKD versus NL, ^#P < 0.05 35-week CKD versus NL, **P < 0.05 35-week CKD + Ca versus CKD.

FIGURE 3

NCX1 and SERCA2a expression are decreased at 35 weeks. (A) Representative images; n = 6 in each normal group, n = 8–9 in each CKD group. (B) Quantification of the images, normalized to β-actin. For all panels: NL = normal (n = 6); CKD = chronic kidney disease (n = 6); CKD + Ca = chronic kidney disease + Ca²⁺ treatment (n = 8). *P < 0.05 versus normal, ^#P < 0.05 versus CKD without calcium treatment.

FIGURE 4

Schematic of predicted mechanism by which altered[Ca²⁺]_i regulation contributes to elevated resting [Ca²⁺]_i. The presence of a variety of uremic toxins in the extracellular environment may either directly or indirectly contribute to the described perturbations in intracellular Ca²⁺ regulation. Elevated SERCA function drives increases in SR Ca²⁺ store capacity. To compensate for SR Ca²⁺ store overload, SR Ca²⁺ is released either by the RyR or by the IP₃R, initiating signaling mechanisms triggering store-operated Ca²⁺ entry through TRPC1 channels. This phenomenon, together with reductions in NCX expression, cause resting [Ca²⁺]_i to become elevated in CKD. TRPC1 = transient receptor potential canonical 1 channel; NCX = Na⁺/Ca²⁺ exchanger; Ca_i = intracellular free Ca²⁺; IP₃R = inositol 1,4,5-trisphosphate receptor; SERCA = sarco/endoplasmic reticulum Ca²⁺ ATPase; RyR = ryanodine receptor; SR = sarcoplasmic reticulum; Ca_SR = SR Ca²⁺.