Shi Y, Melnikov V Y, Schrier R W, Edelstein C L
Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Am J Physiol Renal Physiol. 2000 Sep;279(3):F509-17. doi: 10.1152/ajprenal.2000.279.3.F509.
The interaction between the cysteine proteases calpain and caspases during renal ischemia-reperfusion (I/R) was investigated. An increase in the activity of calpain, as determined by 1) the appearance of calpain-mediated spectrin breakdown products and 2) the conversion of procalpain to active calpain, was demonstrated. Because intracellular calpain activity is regulated by calpastatin, the effect of I/R on calpastatin was determined. On immunoblot of renal cortex, there was a 50-100% decrease of a low molecular weight (LMW) form of calpastatin (41 kDa) after I/R. Calpastatin activity was also significantly decreased after I/R compared with sham-operated rats, indicating that the decreased protein expression had functional significance. In rats treated with the caspase inhibitor, z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-D-DCB), the decrease in both calpastatin activity and protein expression was normalized, suggesting that caspases may be proteolyzing calpastatin. Caspase 3 activity increased significantly after I/R and was attenuated in ischemic kidneys from rats treated with the caspase inhibitor. In summary, during renal I/R injury, there is 1) calpain activation associated with downregulation of calpastatin protein and decreased calpastatin activity and 2) activation of caspase 3. In addition, in vivo caspase inhibition reverses the decrease in calpastatin activity. In conclusion, proteolysis of calpastatin by caspase 3 may regulate calpain activity during I/R injury. Although the protective effect of cysteine protease inhibition against hypoxic necrosis of proximal tubules has previously been demonstrated, the functional significance in ischemic acute renal failure in vivo merits further study.
研究了肾缺血再灌注（I/R）过程中半胱氨酸蛋白酶钙蛋白酶和半胱天冬酶之间的相互作用。通过以下两点确定了钙蛋白酶活性的增加：1）钙蛋白酶介导的血影蛋白降解产物的出现；2）无活性钙蛋白酶向活性钙蛋白酶的转化。由于细胞内钙蛋白酶活性受钙蛋白酶抑制蛋白调节，因此确定了I/R对钙蛋白酶抑制蛋白的影响。在肾皮质的免疫印迹中，I/R后低分子量（LMW）形式的钙蛋白酶抑制蛋白（4 kDa）减少了50-100%。与假手术大鼠相比，I/R后钙蛋白酶抑制蛋白活性也显著降低，表明蛋白表达的降低具有功能意义。在用半胱天冬酶抑制剂z-Asp-2,6-二氯苯甲酰氧基甲基酮（Z-D-DCB）处理的大鼠中，钙蛋白酶抑制蛋白活性和蛋白表达的降低均恢复正常，提示半胱天冬酶可能在蛋白水解钙蛋白酶抑制蛋白。I/R后半胱天冬酶3活性显著增加，在用半胱天冬酶抑制剂处理的大鼠的缺血肾脏中，该活性减弱。总之，在肾I/R损伤期间，存在：（1）钙蛋白酶激活，伴有钙蛋白酶抑制蛋白下调和钙蛋白酶抑制蛋白活性降低；（2）半胱天冬酶3激活。此外，体内半胱天冬酶抑制可逆转钙蛋白酶抑制蛋白活性的降低。总之，半胱天冬酶3对钙蛋白酶抑制蛋白的蛋白水解作用可能在I/R损伤期间调节钙蛋白酶活性。尽管先前已证明半胱氨酸蛋白酶抑制对近端肾小管缺氧坏死具有保护作用，但其在体内缺血性急性肾衰竭中的功能意义值得进一步研究。
