In vivo and in vitro characterization of the canine nephron and urinary proteome
Date
2011
Authors
Brandt, Laura Elizabeth, author
Bohn, Andrea A., advisor
Ehrhart, Eugene J., committee member
Olver, Christine S., committee member
Prenni, Jessica E., committee member
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Abstract
Immunohistochemistry allows the localization of proteins to specific regions of the nephron. This study reports the identification and localization of proteins in situ within normal canine, feline and mouse kidney by immunohistochemistry, maps their distribution and compares results to previously reported findings in other species. The proteins investigated are aquaporin-1, aquaporin-2, calbindin-d28k, glutathione S-transferase-α and Tamm-Horsfall protein. Our findings in the dog are similar to that in other species, and localize aquaporin-1 to the proximal convoluted tubule epithelium, vasa recta endothelium and descending thin limbs, aquaporin-2 to collecting duct epithelium and calbindin-d28k is found within distal convoluted tubule epithelium. Glutathione S-transferase-α has variable expression and is found in renal transitional epithelium only in some individuals, the proximal straight tubules only in some individuals or in both locations in others. Tamm-Horsfall protein localizes to thick ascending limb epithelium. These findings are similar in the cat, with the exception that aquaporin-1 is located in glomerular podocytes, in addition to proximal convoluted tubule epithelium, and glutathione S-transferase-α is found solely within the proximal convoluted tubule within all kidney samples examined. The mouse kidney is almost identical to the dog, but expresses glutathione S-transferase-α in the glomeruli only. Additionally, we successfully adapted techniques employed in human studies to characterize the normal canine urinary proteome. Both soluble proteins and the exosomal fraction of urine were examined. Greater than 500 proteins were identified in this initial study. Many of these proteins are also found in human urine, but large numbers of proteins also appear to be unique to the canine. Software entitled Biological Networks Gene Ontology (BiNGO) was utilized to characterize canine urinary proteins into respective Gene Ontology categories of Cellular Component, Molecular Function and Biological Process. Exosomal proteins are largely derived from an intracellular location, while soluble proteins are comprised of extracellular and membrane proteins. The majority of proteins in both canine urinary fractions are involved in protein binding. Exosomal proteins are also involved in metabolic process and localization, while soluble proteins are linked to specific localization processes. The distribution of canine urinary proteins within GO functional categories is very similar to that of human urine. Known markers of renal disease, such as aquaporin-1, gamma-glutamyl transferase and retinal binding protein were identified via proteomic techniques in the urine of healthy dogs, as were novel biomarkers like fetuin-A and ubiquitin A-52.
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Subject
biomarkers
canine
immunohistochemistry
nephron
renal failure
urinary proteome