Study On The Mechanism Of Renal Injury Induced By The Jellyfish Venom

IntroductionIn recent years coastal waters around the globe, including China, have seen massivejellyfish outbreak. Meanwhile, with the increasing of ocean-related human activities,jellyfish sting has become the most common injuries caused by marine animals. Jellyfishpray and defend themselves by injecting venom through nematocysts, and sting animalsand human. It has been reported that the syndromes such as skin allergy, pain or evencardiopulmonary failure appear to victims after stung by jellyfish. Researches showed thatjellyfish venom may cause severe localized and systemic diseases, and can even causedeath. The increasing of jellyfish sting accidents is causing panic throughout coastalfishermen and tourists.Cyanea capillata has a relative mild toxicity, and has never been reported to causedeath to human, yet it is widely distributed in China and is one of the main jellyfish thatinduce injury in China’s southeast coast. Multi-organ dysfunction emerges on some of thepatients after stung by jellyfish, and our research team has also found previously that thetentacle extract (TE) of Cyanea capillata could induce kidney injury of the model animal.Under the application of TE, large amounts of vacuoles and hyaline substance emerge inthe lumen of the distal tubules and the glomerular capsule of the rat’s renal cortex,accompanied by necrosis of the renal tubular epithelial cells and opening of medullanephrica vascular bed, and further observation with electron microscope reveals thatglomerular filtration membrane has no obvious change while the renal tubular epithelialcells are severely injured. However, the detailed mechanism underlying the kidney injuryis still obscure. Therefore in this project, we take the most damaged renal tubular epithelialcells as our primary investigation target, and explore the mechanism of TE’s toxic effect onkidney, especially the renal tubular epithelial cells.MethodsPart I: Investigation on the mechanism of injury in rat kidney by TEFirst, taking the kidney sample from the delayed jellyfish envenomation syndromemodel and detecting the rat kidney cell’s apoptosis by TUNEL, as well as the Caspase-3expression in kidney by immunohistochemistry. By the aid of the MTT method, we detected the influence of TE of different concentrations on the survival rate of the rat’srenal tubular epithelial cell NRK-52E, and observed the change of cellular morphologyusing the confocal microscope. The Annexin V-FITC/PI double staining was used todetect cellular apoptosis by a flow cytometer. The role the membrane receptor-mediatedsignaling played in kidney cell injury was demonstrated by differential proteins in themembrane screening using two-dimensional electrophoresis and determination ofintracellular calcium using laser scanning confocal microscope. The expression of theapoptosis related molecules were determined by western blotting, and the effect ofmitochondrial pathway on the tubular cell’s damage was illustrated by the detection of thechange of mitochondria membrane potential and reactive oxygen species (ROS) using laserconfocal spectroscopy, as well as by the measurement of MDA using thiobarbituric acidmethod.Part II: Intervention effect of polyethylene glycol (PEG) on kidney cell damageCells were stimulated with TE under the intervention of PEG6000. By the aid of theMTT method we detected the intervention effect of PEG6000on the survival rate of thecell NRK-52E, and observed the cellular morphology using the confocal microscope. TheAnnexin V-FITC/PI double staining was used to detect cellular apoptosis by a flowcytometer. Mitochondria membrane potential and reactive oxygen species (ROS) weredetected using laser confocal spectroscopy. MDA concentration was measured bythiobarbituric acid method.ResultsIn comparison with normal rat kidney, the renal biopsies of DJES appeared massivebrownish-yellow deceased cell and cell debris under tunel staining, and the expression ofCaspase-3increased. The toxic effect of TE on NRK-52E cell is both time-and dosage-dependent, where the change of cellular morphology happened such as shrinking. Moreapoptosis and necrocytosis occurred with higher TE concentration. Differential proteinsin the membrane, before and after the application of TE, weren’t screened out. TEincreased the concentration of intracellular calcium, while some of the regular calciumantagonists could not resist this phenomenon. PEG6000had a significant resistant effecton the increase of intracellular calcium of both NRK-52E and HEK-293.Under the intervention of PEG6000at the concentration25mM, TE did not lead tosignificant morphology change of NRK-25E cells, whose survival rate were also increased. PEG6000was able to reduce apoptosis and necrocytosis as well as the TE-inducedincrease of ROS and MDA level, and maintain the mitochondria membrane potential.ConclusionThe mechanism for the Ca2+overload in renal tubular epithelial cell induced by TE iscellular Ca2+influx, and its pathway is not the known calcium channel but the membranechannels formed by jellyfish venom. The formation of channels gives rise to a huge Na+and Ca2+influx as well as a K+outflow, changing the osmotic pressure and causing lowermitochondria membrane potential, accumulation of ROS, and triggering the mitochondrialpathway of apoptosis.PEG6000can fundamentally intervene the formation of the membrane channels sothat the consequent reactions such as Ca2+overload and ROS accumulation are resisted,hence providing protection for the injury effect of jellyfish venom.