Ding Fangrui, Li Xuejuan, Li Baihong, Guo Jifan, Zhang Yanqin, Ding Jie
Department of Pediatrics, Peking University First Hospital, Beijing, China.
PLoS One. 2016 May 12;11(5):e0155504. doi: 10.1371/journal.pone.0155504. eCollection 2016.
The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus are widely used in the treatment of proteinuria diseases. As the direct target of these drugs, calcineurin has previously been demonstrated to play a role in proteinuria diseases. However, aside from its immune-related effects, the local status of calcineurin in renal inherent cells has not been fully explored in the settings of proteinuria disease and podocyte injury. In this study, calcineurin activity and protein expression in the well-known puromycin aminonucleoside (PAN)-induced podocyte injury model were examined. Interestingly, we found that calcineurin activity was abnormally increased in PAN-treated podocytes, whereas the expression of the full-length 60-kDa calcineurin protein was decreased. This result suggests that there may be another activated form of calcineurin that is independent of the full-length phosphatase. To investigate whether calpain is involved in regulating calcineurin, we exposed PAN-treated podocytes to both pharmacological inhibitors of calpain and specific siRNAs against calpain. Calpain blockade reduced the enhanced calcineurin activity and restored the down-regulated expression of 60-kDa calcineurin. In addition, purified calpain protein was incubated with podocyte extracts, and a 45-kDa fragment of calcineurin was identified; this finding was confirmed in PAN-induced podocyte injury and calpain inhibition experiments. We conclude that calcineurin activity is abnormally increased during PAN-induced podocyte injury, whereas the expression of the full-length 60-kDa calcineurin protein is down-regulated due to over-activated calpain that cleaves calcineurin to form a 45-kDa fragment.
钙调神经磷酸酶抑制剂环孢素A（CsA）和他克莫司被广泛用于治疗蛋白尿疾病。作为这些药物的直接靶点，钙调神经磷酸酶此前已被证明在蛋白尿疾病中起作用。然而，除了其免疫相关作用外，在蛋白尿疾病和足细胞损伤的情况下，肾脏固有细胞中钙调神经磷酸酶的局部状态尚未得到充分研究。在本研究中，我们检测了在著名的嘌呤霉素氨基核苷（PAN）诱导的足细胞损伤模型中钙调神经磷酸酶的活性和蛋白表达。有趣的是，我们发现PAN处理的足细胞中钙调神经磷酸酶活性异常增加，而全长60 kDa钙调神经磷酸酶蛋白的表达却降低。这一结果表明，可能存在另一种独立于全长磷酸酶的钙调神经磷酸酶激活形式。为了研究钙蛋白酶是否参与调节钙调神经磷酸酶，我们将PAN处理的足细胞暴露于钙蛋白酶的药理学抑制剂和针对钙蛋白酶的特异性小干扰RNA（siRNA）中。抑制钙蛋白酶可降低增强的钙调神经磷酸酶活性，并恢复下调的60 kDa钙调神经磷酸酶表达。此外，将纯化的钙蛋白酶蛋白与足细胞提取物一起孵育，鉴定出一个45 kDa的钙调神经磷酸酶片段；这一发现在PAN诱导的足细胞损伤和钙蛋白酶抑制实验中得到了证实。我们得出结论，在PAN诱导的足细胞损伤过程中，钙调神经磷酸酶活性异常增加，而全长60 kDa钙调神经磷酸酶蛋白的表达因钙蛋白酶过度激活而被下调，钙蛋白酶将钙调神经磷酸酶切割形成一个45 kDa的片段。
