Abstract
Available indicators of renal dysfunction (blood urea nitrogen, uric acid) in avian species have been shown to be relatively nonspecific and insensitive, because they are readily affected by diet and hydration status, and they are often elevated late in the disease course after the renal functional reserve has been depleted. One aspect of ongoing studies employing the pigeon (Columba livia) model (n=26) compared disposition curves (Vd) of 99mTc-DTPA (diethylene pentaacetic acid) and 99mTc-MAG3 (mercapto-acetyl-triglycine), two commonly used renal function tracers, to that of the endogenous marker creatine. The calculated Vd for 99mTc-DTPA (5.24±0.67%; n=4) and 99mTc-MAG3 (3.64±0.69%; n=5) were smaller than for creatine (20.4±3.43%, n=8). Our results in the pigeon are consistent with the view that 99mTc-DTPA is filtered by the glomeruli, and 99mTc-MAG3 is secreted in the renal tubules. The short-lived tracers 99mTc-DTPA and 99mTc-MAG3 show great promise for noninvasive real-time glomerular and tubular assessment of renal function in this representative avian species. Further work is required to clarify whether creatine may be reabsorbed in the renal tubules as well.
Introduction
Renal disease remains a clinically challenging diagnosis in avian species. Available markers of renal dysfunction principally consist of those that accumulate in the blood due to reduced clearance or those released into the urine as a result of renal epithelial injury. Previous work in the pigeon and other avian species suggested the potential value of blood urea nitrogen (BUN) and uric acid for renal disease detection. However, both these markers have been shown to be relatively nonspecific for indication of disease because they are readily affected by diet, hydration status, and other factors. In addition, the elevation of these parameters in the blood occurs relatively late in the disease course and only after the renal functional reserve is depleted. The development of improved methods for the assessment of renal disease would greatly aid the diagnostic capabilities of avian clinicians. In addition, the use of dynamic study methods may facilitate the early detection of renal function deficits whenever therapeutic efforts may be most effective.
The present study in the pigeon included the following objectives: 1) characterizing the time course of the renal function tracers, 99mTc-DTPA (diethylene pentaacetic acid) and 99mTc-MAG3 (mercapto-acetyl-triglycine); and 2) estimating the volume of distribution (Vd) of these tracers as compared to the Vd for the endogenous marker creatine. These objectives were undertaken to allow meaningful comparisons of the disposition curves of these two commonly used renal function tracers in the pigeon, and to make a crucial step toward the characterization of creatine disposition in the avian kidney.
Methods
Twenty-six healthy cull pigeons (Columba livia) were fed a standard laboratory pigeon diet and water ad lib for 1 week prior to several renal function studies. For the present study, all animals were randomly selected by treatment and anesthetized with isoflurane (Aerrane, Anaquest, Madison, WI, USA) and oxygen. Once anesthetized, each pigeon had 22–24-ga heparinized catheters (Abbocath, Abbott Labs, North Chicago, IL, USA) placed into both a medial tarsal vein (blood sampling) and a brachial vein (tracer/marker infusion). Each bird was recovered from anesthesia, administered 10 ml of 0.9% saline SC over the dorsum, and maintained in a quiet environment with low light 1.5 hours prior to study.
Pigeons were anesthetized just prior to study with a combination of ketamine (25 mg/kg IM, Ketaset, Fort Dodge Animal Health, Fort Dodge, IA, USA) and xylazine (2 mg/kg IM, TranquiVed, Vetco, St. Joseph, MO, USA). Once anesthetized, each bird was placed directly on the detector surface of the Anger camera (Siemens Orbiter, Siemens AG, Munich, Germany) in dorsal recumbency on a disposable absorbent plastic barrier-backed sheet. Tissue depth was kept constant by careful positioning. A baseline blood sample was drawn from the medial tarsal vein to measure baseline creatine levels. A combination of tracer (99mTc-DTPA [diethylene pentaacetic acid] and 99mTc-MAG3 [mercapto-acetyl-triglycine], Mallinckrodt Medical, St. Louis, MO, USA) and marker (creatine) was suspended in saline. This tracer/marker cocktail was injected IV into the brachial vein over a period of 30–45 seconds for a total volume of 2.85 ml. Images were collected at 1/second and collapsed into 12 1-minute time series composite images using Image J (via download, National Institutes of Health, https://imagej.nih.gov/ij/index.html). During the scanning period, serial small-volume (0.25–0.5 ml) blood samples were collected to assay for both the tracer and the marker.
Regions of interest (ROI) were delimited with the area drawing tool. The mean counts, the count area (of the ROI), and area normalized counts were calculated. ROIs for the “vascular phase,” the “renal phase,” and the “excretory phase” were selected and delimited from the heart, kidney, and cloaca regions, respectively. Preliminary validations included correcting for background; defining the time course of the vascular, renal, and excretory phases; and surveying for intrarenal functional differences. The volume of distribution (Vd) for each tracer and creatine were calculated by previously reported methods.1 Briefly, Vds of the tracers and creatine were estimated from log transformed y intercepts (time=0), bodyweight, and the amount of tracer or creatine given. Creatine was assayed by methods described previously.3
Results
Initial analyses revealed a peak in the vascular phase approximately 2 minutes post-injection for both tracers. The excretory phase was derived from area normalized subtraction of the heart ROI curve from the cloacal ROI curve. For 99mTc-DTPA, the excretory phase (n=7) started at 4.36±0.18 minutes (mean±SE; n=7), whereas for 99mTc-MAG3 it occurred at 3.07±0.41 minutes (n=7). The “renal excretion phase” was selected as the time period from 2–7 minutes post-injection for subsequent analyses.
The calculated Vd for 99mTc-DTPA (5.24±0.67%; n=4) and 99mTc-MAG3 (3.64±0.69%; n=5) were smaller than for creatine (20.4±3.43%, n=8), suggesting that the creatine disappearance curve was at least partially determined by a larger volume of distribution as compared to the tracers used.
Mean slopes of log transformed renal phase disposition curves for the two tracers were significantly different (p=0.034) between the two tracers. Likewise, the overall disposition curves for birds receiving each tracer indicated that 99mTc-MAG3 (n=5) excretion appeared more rapid and had a steeper negative slope than was observed for 99mTc-DTPA (n=5).
Discussion
The short-lived tracers 99mTc-DTPA and 99mTc-MAG3 show great promise for noninvasive real-time assessment of renal function in this representative avian species. Our results in the pigeon support similar findings in the human that 99mTc-DTPA is filtered by the glomeruli, whereas 99mTc-MAG3 is secreted in the renal tubules.2 Based on earlier work (Wimsatt and Steyn, unpublished, 1997), creatine appeared to have an even slower excretion rate than 99mTc-DTPA in the pigeon. Some of this effect is explained by its large volume of distribution; however, further work is required to clarify whether creatine may be reabsorbed in the renal tubules as well.
Acknowledgments
We thank Dr. Phillip Steyn of Colorado State University for contributing to earlier studies upon which the present studies are based, and Dr. Klaus Beyenbach of Cornell University for helpful advice. Funding for this project was provided by a generous grant from the Morris Animal Foundation (DO2ZO-79 to JW).
Literature Cited
1. Beyenbach K.W., and L.B. Kirschner. 1976. The unreliability of mammalian glomerular markers in teleostean renal studies. J. Exp. Biol. 64: 369–378.
2. Durand, E., and A. Prigent. 2002. The basics of renal imaging and function studies. Q. J. Nucl. Med. 46: 249–267.
3. Wimsatt J., R.D. Pearce, S. Nelson, L.T. Shanahan, and L.M. Vap. 2003. Tissue content of novel renal disease markers in pigeons (Columba livia). Proc. Am. Assoc. Zoo Vet. 2003: 317–318.