Studies on the Mechanisms and Kinetics of Apoptosis Induced by Microinjection of Cytochrome c in Rat Kidney Tubule Epithelial Cells (NRK-52E)

Abstract

Recent reports substantiating the role of cytochrome c in the induction of apoptosis led us to examine the kinetics and mechanisms involved in this process as an extension of our ongoing studies of cell injury and cell death. Microinjection of cytochrome c into NRK-52E kidney cells produced rapid apoptosis, which usually began within 30 minutes and reached a maximum of 60 -70% by 3 hours. The changes that occurred included four phases: an initial shrinkage phase, an active phase, a spherical phase, and a necrotic phase. For morphological purposes, the progressive changes were followed by phase-contrast and fluorescence microscopy, transmission and scanning electron microscopy, and time-lapse video microscopy. Cells first showed shrinkage, then displayed multiple pseudopods, which rapidly extended and retracted, giving the cells a bosselated appearance. During this active phase there was chromatin condensation, mitochondria were swollen but retained membrane potential, and the endoplasmic reticulum was dilated. Within 2-4 hours, active-phase cells became spherical and smooth-surfaced but were still alive, the nuclei showed chromatin clumping, the mitochondria underwent high-amplitude swelling but retained membrane potential, the endoplasmic reticulum was highly dilated, and many large apical vacuoles were present. Elevation of [Ca 2؉ ] i was seen at the late spherical phase, shortly before cell death. Pretreatment with the caspase 3 inhibitor (Ac-DEVD-CHO) prevented apoptosis, whereas overexpression of Bcl-2 did not. Apoptosis is an important mechanism of cell death in the kidney during development and after many and diverse injuries.1 As part of our ongoing studies of cell injury and cell death in the kidney, we are exploring the detailed stages and mechanisms of apoptosis and comparing them with those of oncosis. As the prelethal changes of oncosis have been well characterized, we wished to carefully examine the changes of apoptosis before and after cell death. 2,3Cytochrome c release into the cytosol has been implicated as an intermediate event in the initiation of apoptosis after a variety of toxic and other stimuli and injury in several cell types 4 -9 ; it also serves as a useful model, inasmuch as we found that after microinjection, the early phase of apoptosis usually begins within 30 minutes. This means that the progression of changes in single injected cells can be followed by many important techniques. These include the use of time-lapse phase-contrast video microscopy, digital imaging fluorescence, and confocal microscopy to analyze mitochondrial membrane potential, mitochondrial permeability transition, cytosolic calcium concentration [Ca 2ϩ ] i , and cell death, as well as phase-contrast and electron microscopy for morphological characterization.The results clearly defined three principal prelethal stages or phases of apoptosis and characterized the morphology at the light and electron microscope levels. We also showed that ATP is required for the initiation of apoptosis, that the loss of mitocho...

Synchronous progression of calcium transient-dependent beating and sarcomere destruction in apoptotic adult cardiomyocytes

“…These systematic changes were entirely different from those seen in oncotic cardiomyocytes, in which myofibrils were supercontracted and exhibited contraction bands with preserved Z disks. Ca 2ϩ transients appear to be related to the dynamics of apoptosis ubiquitously observed among various cell types (3,9,23,24). These dynamics would be especially apparent in the beating of cells with well-developed myofilament systems, e.g., adult cardiomyocytes, although they would also be reflected in the proteolysis and DNA fragmentation catalyzed by Ca 2ϩ -dependent protease and DNases.…”

“…Video recording of various apoptotic cell types in culture has revealed apoptosis to be a dynamic process that is complete within hours and is characterized by several common features: cellular shrinkage and rounding, budding or blebbing, and apoptotic body formation (3,23,24). These structural changes are accompanied by disassembly of the cytoskeletal and nuclear scaffold as a result of proteolysis catalyzed by caspase family proteases (25,36).…”

Pinocytic loading of cytochrome c into intact cells specifically induces caspase-dependent permeabilization of mitochondria: evidence for a cytochrome c feedback loop

“…In one case, results from non-quantitative, direct fluorescence microscopic examination of cells stained with a fluorescent, potential-sensitive mitochondrial dye were interpreted as indicating that cytosolic cytochrome c had no effect on mitochondrial polarization. 14 In the second case, it was reported that loss of mitochondrial membrane potential (DC mit ) did not occur until immediately before cell death induced by microinjection of cytochrome c. 17 However, the validity of the technique used in this study to measure changes in DC mit (staining cells with rhodamine 123) was questioned in recent reports. 18,19 Thus, it remains uncertain whether, in intact cells, the release of cytochrome c to the cytosol triggers quantitative changes in mitochondrial polarization or other mitochondrial changes.…”

Identification of thrombospondin 1 (TSP-1) as a novel mediator of cell injury in kidney ischemia

“…TSP-1 at 1 µg/ml was added to cultured kidney proximal tubule (NRK) cells and studied at 24 and 48 hours for evidence of apoptosis by cytochrome c release and caspase-3 activity. The NRK-52E normal kidney cell line is derived from the proximal tubule epithelium (PTE) of the rat kidney, is nontumorigenic, retains many morphological and physiological characteristics of PTE, and has been used to mimic changes in vivo (31)(32)(33). Cytochrome c apoptosis detection kit (Alexa Fluor 488 assay; Invitrogen Corp.) was used to perform the apoptosis assay.…”