We have previously reported that the hydroxyl radical combines with creatinine via its hydroxyl adduct, creatol, to form methylguanidine (MG)1. Based on this finding, we propose that measuring creatol or MG is a useful method to estimate hydroxyl radical generation in vivo. Using this method, we have reported that puromycin aminonucleoside (PAN), a substance that induces nephrosis, increases hydroxyl radical generation in rats both in vivo2 as well as in isolated rat hepatocytes1,3.
The increased generation of creatol in isolated rat hepatocytes was observed within one hour3. A role for these reactive oxygen species in aminonucleoside-induced nephrosis based on the effect of their scavengers has been described4. It has also been reported that PAN increases lipid peroxide of many tissues, including rat kidney and liver5. With this in mind, we looked for increased reactive oxygen species in isolated glomeruli. However, the sensitivity when using creatol or MG was too low for such detection. Because the imaging of hydroperoxides in perfused rat liver6 as well as in cultured cells7 gave useful information concerning the distribution of hydroperoxides and its variation in time, we attempted to image hydroperoxides in glomeruli stimulated by PAN in vivo and in vitro.
METHODS
Hydroperoxides including lipid peroxides were imaged using dichlorofluorescein diacetate (DCFH-DA). DCFH-DA enters into cells and makes dichlorofluorescein, which fluoresces when reacted with hydroperoxides and/or hydrogen peroxide6,7,8.
The fluorescence at an excitation wavelength of 464 nm was detected using a highly sensitive photon detection video camera (VIM camera, C2400-20; Hamamatsu Photonics, Hamamatsu, Japan) with a 150 W Xe lamp and an emission wavelength above 500 nm. This is designed to detect very fine fluorescence from the connected microscope. Two kinds of glomeruli were examined: one is the glomerulus stimulated by PAN in vivo and another is that stimulated in vitro. In the initial studies, glomeruli of male Wistar rats were collected by a sieving method on the seventh day after the intraperitoneal injection of PAN (100 mg/kg body wt). These were incubated in 50 mM Krebs-Henseleite improved 1 buffer containing 5 mM DCFH-DA. In later experiments, the glomeruli obtained by a sieving method were incubated in Krebs-Henseleite bicarbonate buffer with or without PAN or phorbol myristate acetate (PMA). In this case, glomeruli were collected from male Wistar rats at 30 minutes after the intravenous injection of DCFH-DA (1.8 mg/kg body wt) to maintain uniformity of the glomerular samples.
RESULTS
Imaging of fluorescence from the glomeruli isolated from rats stimulated by puromycin aminonucleoside in vivo
It was previously reported that thiobarbituric acid reactive substances (TBARS), a product of oxidation in rat kidney, increases one day after a PAN injection, and the increase continues up to the seventh day5. Initially, imaging hydroperoxides in glomeruli from the nephrotic rats was unsuccessful. On the seventh day, however, the fluorescence was very strong Figure 1b compared with that of glomeruli from control rat Figure 1a. The images of fluorescence captured by moving the camera focus are comparable to the picture of galaxies obtained through a telescope.
Figure 1.
Images of hydroperoxides of isolated glomeruli obtained from a normal Wistar rat (A) and a nephrotic rat (B) seven days following an injection of puromycin aminonucleoside (PAN).
Full figure and legend (54K)Imaging of fluorescence from isolated rat glomeruli stimulated by puromycin aminonucleoside in vitro
Two localized points of fluorescence appeared in every glomerulus after 15 minutes of incubation with 1.9 mM PAN Figure 2a. The images after 40 Figure 2b and 75 minutes Figure 2c showed that the increased fluorescence depends on the incubation period and the fluorescence spreads throughout the glomerulus, giving an image similar to that of the glomeruli of the rats rendered nephrotic by PAN in vivo. When glomeruli were incubated with PMA, two points giving very strong fluorescence were observed Figure 2d, but the fluorescence did not spread throughout the glomeruli. The fluorescence produced by PMA was too strong to detect by VIM camera, requiring a decrease in the sensitivity of the camera. The glomeruli incubated without these stimulants did not give any image in contrast to background, even in the presence of a VIM camera with increased sensitivity.
Figure 2.
Images of hydroperoxides of isolated rat glomeruli stimulatedin vitro by puromycin aminonucleoside (PAN). Glomeruli were obtained after the intravenous injection of DCFH-DA in normal male Wistar rat. The images were made from glomeruli at 15 (A), 40 (B), and 75 minutes (C) after incubation with PAN. The image of glomeruli at 30 minutes following the addition of PMA is shown in (D).
Full figure and legend (46K)DISCUSSION
This may be the first report showing images of hydroperoxides in rat glomeruli stimulated by PAN in vivo and in vitro. Images from the glomeruli stimulated by PAN in vitro confirmed that it took only 15 minutes to increase the reactive oxygen species. This rapid response of glomeruli to PAN supports our previous reports concerning isolated rat hepatocytes1,3. In the current study, we obtained an image of glomeruli isolated from nephrotic rats on the seventh day following PAN injection. In this study, we did not try to look earlier, however, an image similar to that on the seventh day should be demonstrable earlier because an increased level of TBARS in many rat organs, including liver and kidney, was observed from the first day5.
We have also reported that protein kinase C (PKC) activation increases hydroxyl radical generation in hepatocytes9, and the activation of PKC in rat kidney was observed after 45 minutes of PAN injection in vivo (abstract; Aoyagi et al, Nephrology 1:S387, 1997). However, the increased hydroperoxides of glomeruli on the seventh day may be induced by the toxicity of lipid peroxides.
Concerning the different pictures obtained between PAN and PMA, the difference may be related to the different mechanisms of PKC activation. PMA activates PKC directly and generates reactive oxygen in inflammatory cells in amounts 1000 times that of tissue cells. However, neither the direct activation of PKC nor activation of inflammatory cells by PAN has ever been reported. Therefore, the mechanism of activation of PKC related to the generation of reactive oxygen species by PAN could be different from that of PMA. This concept is supported in the great difference in the strength of fluorescence that we observed.
This article presents a new technique for the investigation of intracellular reactive oxygen generation in isolated glomerular cells.
References
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