Simultaneous Determination of Everolimus, Sirolimus, Tacrolimus, and Cyclosporine-A by Mass Spectrometry.

Measurement of immunosuppressive drug concentrations cyclosporine A (CyA), tacrolimus (TAC), sirolimus (SIR), and everolimus (EVE) in blood is an important application of therapeutic drug monitoring. These immunosuppressive agents are used in combined regimens and nowadays the liquid chromatography and tandem mass spectrometry is the best option for simultaneous analysis of these drugs in one short run. We developed an liquid chromatography and tandem mass spectrometry methodology in-house to measure the combination of immunosuppressants in a single blood sample from transplant patients in Brazil. We analyzed 235 combinations of 4 immunosuppressive drugs in patient blood to validate this study. The measuring ranges were 9 to 1000 ng/mL for CyA and 2 to 50 ng/mL for TAC, SIR, and EVE. Accuracy of the method was between 83.87% and 126.6% (coefficient of determination [r2] > 0.995). Validation of variation was ≤15% for lower limit of quantification. In our analysis 20% of patients treated with EVE showed concentration range of 6 to 6.9 ng/mL, 28% of patients treated with SIR showed a concentration range of 4 to 4.9 ng/mL to TAC, 22% of patients showed concentration range of 5 to 5.9 ng/mL, and finally 50% of patients treated with CyA showed concentration range of 20 to 30 ng/mL. Our routine laboratory was able to implement this new methodology in-house to measure simultaneous CyA, TAC, SIR, and EVE in a single blood sample from transplant patients with a sensibility and rapid quantification analysis.

Both aldosterone and spironolactone can modulate the intracellular ACE/ANG II/AT1 and ACE2/ANG (1-7)/MAS receptor axes in human mesangial cells.

The kidney is an important target of the renin-ANG-aldosterone system (RAAS). To date, several studies have demonstrated the existence of a local RAAS in various tissues, including the renal tissue. The mineralocorticoid aldosterone is known to play a critical role in the classical RAAS; however, its effect on mesangial cells (MCs) remains to be elucidated. Based on this, our aim was to investigate whether aldosterone stimulation can modulate the intracellular RAAS of immortalized human MCs by evaluating ANG-converting enzyme (ACE)/ANG II/ANG II receptor type 1 (AT1) and ANG-converting enzyme 2 (ACE2)/ANG (1-7)/MAS receptor axes. To realise this, protein expression, enzyme activity, and immunofluorescence were performed under aldosterone stimulation and in the presence of the mineralocorticoid receptor (MR) antagonist spironolactone (SPI). We observed that high doses of aldosterone increase ACE activity. The effect of aldosterone on the catalytic activity of ACE was completely abolished with the pretreatment of SPI suggesting that the aldosterone-induced cell injuries through ANG II release were attenuated. Aldosterone treatment also decreased the expression of MAS receptor, but did not alter the expression or the catalytic activity of ACE 2 and ANG (1-7) levels. Spironolactone modulated the localization of ANG II and AT1 receptor and decreased ANG (1-7) and MAS receptor levels. Our data suggest that both aldosterone and the MR receptor antagonist can modulate both of these axes and that spironolactone can protect MCs from the damage induced by aldosterone.