Quantitative cytoplasmic TUNEL: the method to measure apoptosis and necrosis coexisting in a single liver or kidney cell

Apoptosis in solid organs often develops to and overlaps with necrosis, presenting so called “aponecrosis.” Methods for simultaneous quantification of apoptosis and necrosis in a single cell do not exist. We tested whether quantitative fluorescent TUNEL assay that measures 3'OH DNA termini produced by endogenous endonucleases in both apoptosis and necrosis, and in particular, the quantification of the cytoplasmic fraction of TUNEL that is a sign of necrosis, can be applied for apoptosis and necrosis measurement in single cells in two in vivo models: renal ischemia‐reperfusion (IR) in rats and liver acetaminophen (APAP)‐induced injury in mice. Our data showed that renal IR results in full spectrum of tubular epithelial cells undergoing apoptosis conversion to complete necrosis (~50% of total TUNEL leaked to cytoplasm), with the maximum necrosis concentrating in cortico‐medullary junction of the kidney. In centrilobular hepatocytes, the TUNEL‐positive DNA also significantly leaked to cytoplasm, but it reached a maximum of 22% of total TUNEL, indicating less secondary necrosis compared to the kidney. Overall, these data demonstrated that measurement of nuclear and cytoplasmic TUNEL is a useful method for the assessment of coexisting apoptosis and necrosis in individual cells in vivo and a tool to measure progression of kidney or liver injury. The study was supported by NIH/NIDDK grant to A.G.B, and VA Merit Review grants to A.G.B. and S.V.S.