Abstract

Marine mammal medicine is hampered by a shortcoming of markers that can diagnose or predict disease outcomes. One such shortcoming revolves around kidney injury, which is primarily diagnosed by elevated serum creatinine concentration. Serum creatinine is a marker of filtration and cannot differentiate between pre-renal azotemia and tubular injury, and lags behind kidney injury 24 to 48 hours, thus making this test less sensitive at detecting acute kidney injury. Urine proteins have been popular targets for biomarker exploration in both human and veterinary studies in an effort to estimate renal injury in lieu of traditional filtration markers. The use of urine biomarkers in the field of marine mammal medicine has been slower to develop, perhaps due to the fact that urine proteins from marine mammals are less well-described. We utilized proteomics to identify specific urine proteins as candidate markers of renal injury in California sea lions (CSLs: Zalophus californianus) with Leptospirosis. This disease, caused by infection with pathogenic species in the genus Leptospira, is one of the most common reasons for CSLs to strand and be treated at The Marine Mammal Center (TMMC).^1,2 Infection is associated with interstitial renal disease ranging from subclinical to severe. For this study, we analyzed urine proteins from CSLs divided into two groups: Leptospirosis Group CSLs (n=11) were categorized as having active Leptospira infection with associated renal disease and were being treated at TMMC; Non-Leptospirosis Group CSLs (n=8) were categorized as apparently healthy, with no prior or current Leptospira infection and had been sampled in the wild. Both groups were matched for sex and age class. Tryptic peptides were separated by nanoflow chromatography and analyzed using a Thermo Orbitrap Fusion Lumos Tribrid mass spectrometer. Spectra were searched against a combined Pacific walrus (Odobenus rosmarus divergens) and Weddell seal (Leptonychotes weddellii) proteome to make identifications via Mascot (v2.6.0; Matrix Science). Identification confidence thresholds were set using Scaffold (v. 4.7.2; Proteome Software, Portland, OR, USA), with a minimum protein threshold of 1% False Discovery Rate (FDR), and 1% FDR at the peptide level. Experiment-wide grouping was used, which resulted in 2694 protein families identified. Weighted spectral counts were exported from Scaffold for downstream analysis. For differential analysis, a two-sided Wilcoxon rank sum test was used, and false discovery rate for multiple hypothesis testing was estimated using the method of Benjamini and Hochberg (BH). Of these identified protein groups, 316 were differentially abundant (BH corrected p<0.05) with 98 being increased in the Leptospirosis group and 218 being decreased. Established tubular injury markers such as Neutrophil Gelatinase Lipocalin and Osteopontin were greater than 15-fold elevated and nearly exclusively identified in CSLs with Leptospirosis kidney injury. Inter-Alpha-Trypsin Inhibitor Heavy Chain H1 showed the greatest positive fold change (35-fold) compared to CSLs without Leptospirosis. Based on these results, analysis of urine proteins for the detection of kidney injury in CSLs offers promise for selection and validation of candidate biomarkers for identification of renal injury in marine mammals.

Acknowledgements

The authors would like to acknowledge volunteers and staff at The Marine Mammal Center, Sausalito, CA, the Marine Mammal Laboratory, NOAA, NMFS, Oregon and Washington Departments of Fish and Wildlife, and specifically Renee Galloway at the Centers for Disease Control and Prevention and David Wu at the Hollings Marine Laboratory. This work was supported in part by grants through the National Science Foundation (NSF OCE-1335657) and the Department of Defense Environmental Research Program (SERDP RC-2635).

* Presenting author

Literature Cited

1.  Greig DJ, et al. (2005). A decade of live California sea lion (Zalophus californianus) strandings along the central California coast: causes and trends, 1991–2000. Aquatic Mammals. 33(1):11–22.

2.  Gulland FMD, et al. (1996). Leptospirosis in California sea lions (Zalophus californianus) stranded along the central California coast, 1981–1994. Journal of Wildlife Diseases. 32(4):572–580.