Kaushal G P, Singh A B, Shah S V
Division of Nephrology, University of Arkansas for Medical Sciences, Little Rock, USA.
Am J Physiol. 1998 Mar;274(3):F587-95. doi: 10.1152/ajprenal.1998.274.3.F587.
In the present study, we demonstrate that rat kidney contains caspase activity that was markedly inhibited by specific peptide inhibitors of caspases but not by inhibitors of Ser, Cys, Asp, or metalloproteinases. Using primers based on the nucleotide sequence of known members of Ced-3/interleukin-1 beta-converting enzyme (ICE) family from human origin, we have identified by reverse-transcription (RT) polymerase chain reaction (PCR) analyses that rat kidney transcribes the genes for caspase-1 (ICE), caspase-2 (Nedd2), caspase-3 (CPP32), and caspase-6 (Mch2). RT-PCR products, when subcloned and sequenced, provided full-length cDNAs for ICE (1,209 bp) and CPP32 (786 bp) and partial cDNA products for Mch2 (561 bp) and Nedd2 (811 bp). The sequence analysis of the caspase cDNAs showed conserved catalytic site QACRG as well as Asp cleavage site. Rat kidneys subjected to ischemia-reperfusion injury revealed differential expression of caspases with marked increase in CPP32 and ICE mRNA and proteins during reperfusion, transient increase in Nedd2 mRNA and proteins during ischemia and the early period of reperfusion, and little change in Mch2 expression during the ischemia or reperfusion period. The altered expression suggests that caspases may act in concert in a cascade and may play an important role in ischemic acute renal failure.
在本研究中，我们证明大鼠肾脏含有半胱天冬酶活性，该活性被半胱天冬酶的特异性肽抑制剂显著抑制，但不受丝氨酸、半胱氨酸、天冬氨酸或金属蛋白酶抑制剂的抑制。利用基于人类来源的Ced-3/白细胞介素-1β转换酶（ICE）家族已知成员核苷酸序列的引物，我们通过逆转录（RT）聚合酶链反应（PCR）分析鉴定出大鼠肾脏转录半胱天冬酶-1（ICE）、半胱天冬酶-2（Nedd2）、半胱天冬酶-3（CPP32）和半胱天冬酶-6（Mch2）的基因。RT-PCR产物经亚克隆和测序后，提供了ICE（1209 bp）和CPP32（786 bp）的全长cDNA，以及Mch2（561 bp）和Nedd2（811 bp）的部分cDNA产物。半胱天冬酶cDNA的序列分析显示了保守的催化位点QACRG以及天冬氨酸切割位点。遭受缺血再灌注损伤的大鼠肾脏显示半胱天冬酶的表达存在差异，再灌注期间CPP32和ICE的mRNA及蛋白显著增加，缺血期间及再灌注早期Nedd2的mRNA及蛋白短暂增加，而缺血或再灌注期间Mch2的表达变化不大。表达的改变表明半胱天冬酶可能以级联方式协同作用，并可能在缺血性急性肾衰竭中起重要作用。
