Isoform-specific inhibition of calcineurin to prevent nephrotoxicity

描述(申请人提供)：L-钙调神经磷酸酶是一种重要的调节酶，在许多细胞过程中发挥作用，包括T细胞信号转导。环孢素和FK506等抑制钙调神经磷酸酶(Cis)的药物在临床上可用于抑制器官移植后的免疫系统。不幸的是，钙调神经磷酸酶的抑制也会影响免疫系统以外的组织，长期使用顺铂往往会产生治疗上的限制性副作用，包括肾毒性和心血管疾病。我们实验室的工作重点是钙调神经磷酸酶在肾脏中作用的特异性。有趣的是，当我们研究缺乏钙调神经磷酸酶催化亚单位α亚基的小鼠和缺乏β亚基的小鼠时，我们发现了非常不同的效果。也就是说，丢失阿尔法会严重损害肾脏的发育，改变细胞周期调节，并导致基质产生增加--这与氯肾毒性相一致，而缺乏β亚型的小鼠则没有明显的肾脏损伤迹象。此外，我们还发现，α/-小鼠会患上高脂血症，这表明钙调神经磷酸酶的作用与心血管疾病的发生有直接联系。最后，缺乏β亚型的小鼠此前已被描述为具有类似免疫抑制的表型，而阿尔法缺失型动物的T细胞功能仍然可以被环孢素抑制。这些小鼠模型清楚地表明，钙调神经磷酸酶亚型具有不同的功能，这些功能在移植环境中特别相关。抑制α亚型会导致肾毒性和高脂血症，而抑制β亚型则会抑制T细胞信号转导。因此，我们假设选择性抑制β亚型将导致免疫抑制，毒副作用较少。为了验证这一假设，有必要建立一个小鼠模型，使我们能够仔细比较选择性丢失钙调神经磷酸酶亚型的影响。由于α/-小鼠的寿命只有几周，目前还不可能进行这样的研究。然而，基因操作的最新进展提供了创新的工具来产生可诱导的、同工型特异性的基因敲除小鼠。我们将创建这些小鼠，并表征每种亚型的可诱导性丢失在免疫抑制和肾毒性/心血管副作用中的作用。使用一个特征明确的皮肤移植模型，然后我们将检验这一假设，即选择性抑制钙调神经磷酸酶异构体会导致免疫抑制，副作用更少。

DESCRIPTION (provided by applicant): L-Calcineurin is an important regulatory enzyme that functions in many cellular processes including T cell signal transduction. Drug such as cyclosporin and FK506 that inhibit calcineurin (CIs) are clinically useful to suppress the immune system following organ transplantation. Unfortunately, inhibition of calcineurin also affects tissues other than the immune system and long-term use of CIs often produces therapeutically-limiting side-effects including nephrotoxicity and cardiovascular disease. Work in our laboratory is focused on specificity of calcineurin action in the kidney. Interestingly, when we examined mice that lack the alpha isoform of the catalytic subunit of calcineurin and mice that lack the beta isoform, we find very different effects. Namely, loss off alpha severely impairs development of the kidney, alters cell cycle regulation, and results in increased matrix production - features consistent with Cl nephrotoxicity while mice lacking the beta isoform lack signs of significant renal damage. Moreover, we find that alpha-/- mice develop hyperlipidemia, demonstrating a direct connection between calcineurin action and development of cardiovascular disease. Finally, mice lacking the beta isoform have been previously described to have an "immune-suppressed"-like phenotype while T cell function in alpha null animals can still be suppressed with cyclosporin. These mouse models clearly demonstrate that calcineurin isoforms have distinct functions that are particularly relevant in the transplant setting. Inhibition of the alpha isoform produces nephrotoxicity and hyperlipidemia while inhibition of the beta isoform results in suppression of T cell signaling. Therefore, we hypothesize that selective inhibition of the beta isoform will result in immune suppression with fewer toxic side-effects. To test this hypothesis, it will be necessary to generate a mouse model that will allow us to carefully compare the effects of selective loss of calcineurin isoforms. Since alpha-/- mice live only a few weeks, it is not currently possible to perform such studies. However, recent advances in gene manipulations provide innovative tools to generate inducible, isoform-specific knockout mice. We will create these mice and characterize the effect of inducible loss of each isoform in immune suppression and nephrotoxic / cardio- vascular side- effects. Using a well-characterized skin transplant model, we will then test the hypothesis that selective inhibition of calcineurin isoforms results in immune suppression with fewer side-effects.