Localization of Megalin Along Zebrafish Lateral Line; Implications for Ototoxicity of Nephrotoxic Drugs

Megalin (gp330, Low Density Lipoprotein Receptor-related Protein 2 (LRP2)) is an endocytic receptor most abundantly located in the renal proximal tubule and the epithelial cells of the inner ear in mammals. Megalin acts as a receptor for aminoglycosides and other polybasic drugs that cause nephrotoxic and ototoxic effects in mammals. The presence of megalin has been shown in Zebrafish embryos however, its potential role in the ototoxic response to aminoglycosides and polymyxins has only recently been suggested through work by this lab. The fact that zebrafish hair cells are both physiologically and morphologically similar to that of human hair cells, along with our studies, indicate that Megalin is likely located  in or around the Zebrafish hair cells of the lateral line. On being exposed to aminoglycosides and polymyxins, zebrafish endured a loss of fluorescently-labeled hair cells, corresponding to cell death and an ototoxic effect, supporting the aforementioned theory.  Furthermore, the application of known Megalin binding agents led to partial protection of drug induced zebrafish hair cell death. It is thus the purpose of this study to determine whether Megalin is located in or around the hair cells on the lateral line of zebrafish as well as further investigate the effectiveness of protective agents in preventing hair cell death and loss of fluorescence along the zebrafish lateral line. Only partial protection was seen with the megalin binding agent regardless of treatment scenario. Cephalosporins, known nephrotoxic drugs that do not go through megalin, were tested on the fish and no loss of fluorescence was seen up to 100 µM, further supporting the conclusion that megalin plays a key role in  the mechanism through which nephrotoxic drugs illicit an ototoxic effect in zebrafish.

Drugs that are known to go through the megalin pathway could conceivably be screened in the Zebrafish by using the ototoxic effects as a potential predictor for  nephrotoxicity in mammals.

Zebrafish at 5 days post fertilization (dpf) (when the hair cells are mature) were labeled with an anti-megalin antibody and imaged using confocal and fluorescence microscopy. Megalin antibody staining on the fish showed patterns closely resembling the neuromast structures seen with fluorescent YoPro1 labeling. These results lead to the conclusion that Megalin is located in the neuromast structure of the hair cells along the lateral line of the zebrafish. 4 dpf zebrafish were treated with a concentration response of the known nephrotoxic polymyxins and gentamicin with or without poly-L-asparagine, a Megalin binding agent for  one day. The fish were then labeled with the fluorescent dye YoPro1 and imaged using fluorescence microscopy to look for a loss of hair cell fluorescence versus the controls.