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Long-term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients.

López E, Berna-Erro A, Bermejo N, Brull JM, Martinez R, Garcia Pino G, Alvarado R, Salido GM, Rosado JA, Cubero JJ, Redondo PC - J. Cell. Mol. Med. (2013)

Bottom Line: Blood samples were drawn from healthy volunteers and kidney transplant patients long-term medicated with rapamycin: sirolimus and everolimus.Our results indicate that rapamycin evokes a biphasic time-dependent alteration in calcium homeostasis and function in platelets from kidney transplant patients under rapamycin regime, as demonstrated by the reduction in granule secretion observed and subsequent impairment of platelet aggregation in these patients compared with healthy volunteers.Platelet count was also reduced in these patients, thus 41% of patients presented thrombocytopenia.

View Article: PubMed Central - PubMed

Affiliation: Cell Physiology Research Group, Department of Physiology, University of Extremadura, Cáceres, Spain.

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Related in: MedlinePlus

Calcium homeostasis in patients under sirolimus and everolimus medication. Fura-2-loaded platelets isolated from healthy (black solid lines) and patients treated with sirolimus (grey lines) or everolimus (black-doted lines), were suspended in Ca2+-free HBS medium (100 μM EGTA was added; arrowheads) and subsequently stimulated either with Thr (A), ADP (B) and THBQ (C) for 3 min., followed by addition of 300 μM of CaCl2 to the extracellular medium to initiate calcium entry. Representative calcium signals of patients belonging to group II are plotted and histograms on the right hand side, represent calcium release and entry as percentage of control of patients treated with sirolimus (n = 6 each group) and everolimus (n = 4 each group) and medicated during less than 24 (I), 24–36 (II), 36–60 (III) and over 60 months (IV). *, ** and ***, represents P < 0.05, <0.01 and <0.01 compared with healthy individuals respectively.
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fig01: Calcium homeostasis in patients under sirolimus and everolimus medication. Fura-2-loaded platelets isolated from healthy (black solid lines) and patients treated with sirolimus (grey lines) or everolimus (black-doted lines), were suspended in Ca2+-free HBS medium (100 μM EGTA was added; arrowheads) and subsequently stimulated either with Thr (A), ADP (B) and THBQ (C) for 3 min., followed by addition of 300 μM of CaCl2 to the extracellular medium to initiate calcium entry. Representative calcium signals of patients belonging to group II are plotted and histograms on the right hand side, represent calcium release and entry as percentage of control of patients treated with sirolimus (n = 6 each group) and everolimus (n = 4 each group) and medicated during less than 24 (I), 24–36 (II), 36–60 (III) and over 60 months (IV). *, ** and ***, represents P < 0.05, <0.01 and <0.01 compared with healthy individuals respectively.

Mentions: Correlation analysis performed in kidney transplant patients, revealed that sirolimus administration for long periods might alter calcium entry, being particularly affected the group II of patients (medicated for 24–36 months; Table 1), although trough levels of sirolimus are unlikely the key factors. As shown in Figure 1, fura-2-loaded platelets from patients and healthy individuals were suspended in a Ca2+-free HBS medium (100 μM EGTA was added), and were stimulated for 3 min. with thrombin (Thr; 0.1 U/ml; Fig. 1A) or ADP (10 μM; Fig. 1B) and then 300 μM CaCl2 was added to the extracellular medium to initiate calcium entry. Our results indicate that both Ca2+ release and entry in response to Thr were altered in most of the groups analysed, being most evident in group II of patients compared with healthy individuals (see Fig. 1A and 1B, where sirolimus reduced in Ca2+ entry evoked by Thr in a 59.8 ± 14.1% (P < 0.01; n = 6)). The effect of sirolimus on ADP-evoked Ca2+ mobilization was not so evident as presented for Thr, but it resulted in a small and time-dependent increase in Ca2+ release among the different patient groups as compared with healthy individuals. Meanwhile, reduced Ca2+ entry in these groups was observed, and despite this difference was not statistically significant a clear tendency was found [32.9 ± 28.0% (P > 0.05; n = 4) in group II]. The different effect on Thr- and ADP-evoked Ca2+ signals might be explained because of the fact that ADP releases Ca2+ from the dense tubular system (DTS; similar to the endoplasmic reticulum in other cells) and Thr mobilizes calcium from the DTS and the acidic stores 36.


Long-term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients.

López E, Berna-Erro A, Bermejo N, Brull JM, Martinez R, Garcia Pino G, Alvarado R, Salido GM, Rosado JA, Cubero JJ, Redondo PC - J. Cell. Mol. Med. (2013)

Calcium homeostasis in patients under sirolimus and everolimus medication. Fura-2-loaded platelets isolated from healthy (black solid lines) and patients treated with sirolimus (grey lines) or everolimus (black-doted lines), were suspended in Ca2+-free HBS medium (100 μM EGTA was added; arrowheads) and subsequently stimulated either with Thr (A), ADP (B) and THBQ (C) for 3 min., followed by addition of 300 μM of CaCl2 to the extracellular medium to initiate calcium entry. Representative calcium signals of patients belonging to group II are plotted and histograms on the right hand side, represent calcium release and entry as percentage of control of patients treated with sirolimus (n = 6 each group) and everolimus (n = 4 each group) and medicated during less than 24 (I), 24–36 (II), 36–60 (III) and over 60 months (IV). *, ** and ***, represents P < 0.05, <0.01 and <0.01 compared with healthy individuals respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3822816&req=5

fig01: Calcium homeostasis in patients under sirolimus and everolimus medication. Fura-2-loaded platelets isolated from healthy (black solid lines) and patients treated with sirolimus (grey lines) or everolimus (black-doted lines), were suspended in Ca2+-free HBS medium (100 μM EGTA was added; arrowheads) and subsequently stimulated either with Thr (A), ADP (B) and THBQ (C) for 3 min., followed by addition of 300 μM of CaCl2 to the extracellular medium to initiate calcium entry. Representative calcium signals of patients belonging to group II are plotted and histograms on the right hand side, represent calcium release and entry as percentage of control of patients treated with sirolimus (n = 6 each group) and everolimus (n = 4 each group) and medicated during less than 24 (I), 24–36 (II), 36–60 (III) and over 60 months (IV). *, ** and ***, represents P < 0.05, <0.01 and <0.01 compared with healthy individuals respectively.
Mentions: Correlation analysis performed in kidney transplant patients, revealed that sirolimus administration for long periods might alter calcium entry, being particularly affected the group II of patients (medicated for 24–36 months; Table 1), although trough levels of sirolimus are unlikely the key factors. As shown in Figure 1, fura-2-loaded platelets from patients and healthy individuals were suspended in a Ca2+-free HBS medium (100 μM EGTA was added), and were stimulated for 3 min. with thrombin (Thr; 0.1 U/ml; Fig. 1A) or ADP (10 μM; Fig. 1B) and then 300 μM CaCl2 was added to the extracellular medium to initiate calcium entry. Our results indicate that both Ca2+ release and entry in response to Thr were altered in most of the groups analysed, being most evident in group II of patients compared with healthy individuals (see Fig. 1A and 1B, where sirolimus reduced in Ca2+ entry evoked by Thr in a 59.8 ± 14.1% (P < 0.01; n = 6)). The effect of sirolimus on ADP-evoked Ca2+ mobilization was not so evident as presented for Thr, but it resulted in a small and time-dependent increase in Ca2+ release among the different patient groups as compared with healthy individuals. Meanwhile, reduced Ca2+ entry in these groups was observed, and despite this difference was not statistically significant a clear tendency was found [32.9 ± 28.0% (P > 0.05; n = 4) in group II]. The different effect on Thr- and ADP-evoked Ca2+ signals might be explained because of the fact that ADP releases Ca2+ from the dense tubular system (DTS; similar to the endoplasmic reticulum in other cells) and Thr mobilizes calcium from the DTS and the acidic stores 36.

Bottom Line: Blood samples were drawn from healthy volunteers and kidney transplant patients long-term medicated with rapamycin: sirolimus and everolimus.Our results indicate that rapamycin evokes a biphasic time-dependent alteration in calcium homeostasis and function in platelets from kidney transplant patients under rapamycin regime, as demonstrated by the reduction in granule secretion observed and subsequent impairment of platelet aggregation in these patients compared with healthy volunteers.Platelet count was also reduced in these patients, thus 41% of patients presented thrombocytopenia.

View Article: PubMed Central - PubMed

Affiliation: Cell Physiology Research Group, Department of Physiology, University of Extremadura, Cáceres, Spain.

Show MeSH
Related in: MedlinePlus