Abstract

Diagnosis of mild to moderate acute or active renal disease remains clinically challenging in avian species, yet early intervention can provide the best outcome. Available markers of avian renal dysfunction principally consist of those that accumulate in the blood due to reduced clearance, and those released into the urine as a result of renal tubular epithelial injury. Previous work in the pigeon and other avian species has suggested the potential value of BUN and uric acid for the detection of renal injury.¹ However, both of these markers are variously influenced by diet, hydration status, and metabolic state. Likewise, the sensitivity of these markers is limited when significant normal renal tissue exists, as often occurs early in the disease process. Preliminary work suggested that gamma glutamyl transferase (GGT) and N-acetyl-β-D- glucosaminidase (NAG) increased in the urine in response to an acute renal insult; however, the former compound was relatively labile (Wimsatt et al. 1995, unpublished data). Creatine is the preponderant metabolite of the energy substrate creatine phosphate excreted from avian muscle. Thus, depending on its distribution in the body, and on its renal disposition, it could serve as a renal disease marker similar to creatinine in mammals. The objective of the present study was to characterize the histopathologic changes associated with gentamicin toxicity in the pigeon, and correlate these pathologic changes with blood plasma levels of uric acid, NAG and creatine, and urine levels of NAG and creatine.

Methods

All procedures were approved by the institutional animal care and use committee. Nineteen culled breeder homing pigeons (n=19; 5 controls and 14 treated) in excellent health were donated to the project, and were placed on a specially formulated semi-liquid 8% protein enteral diet. The prepared enteral formula provided complete maintenance nutrition and hydration (dietary water) when fed on a body weight basis, and was delivered in divided twice daily oral tube feedings. Prior to the study, pigeons were fed on this diet 1 wk, while their body weight, and stool/urate volume and consistency, blood parameters and overall demeanor and health were monitored. During the experimental period, 50 mg/kg gentamicin (Schering, 50 mg/ml) was intramuscularly delivered in twice daily doses. Birds were blood sampled and plasma (Na heparin) was collected before gentamicin treatment started, and on the last study day of gentamicin treatment, prior to humane euthanasia. Fixed tissues were submitted to treatment-blinded histopathologic examination by a specialty boarded pathologist for renal histopathologic tissue scoring. Tissue scores were as follows: 0=normal tissue, 1=mild changes, 2=moderate changes, and 3=severe changes. Parameters scored consisted of proximal tubule (PT) degeneration, PT inflammation, PT necrosis, distal tubule (DT) degeneration, DT inflammation, DT necrosis, and (intraluminal) urate deposition.

For plasma and urine analyses, an original Boehringer-Mannheim creatinine procedure was modified and validated to measure creatine using a Hitachi 917 clinical chemistry autoanalyzer for high throughput, as previously reported.² NAG and uric acid (UA) were assayed using standard commercial kits (Roche) validated for avian species, and run on the same analyzer. Statistical analysis was performed using a Kruskal-Wallis nonparametric ANOVA (tissue scores; this method ranks, and does not require equidistant scoring intervals) and Student’s independent sample T Tests, employing the Bonferroni correction (plasma and urine markers), employing SPSS version 11.0 for Windows XP. In all cases, α=0.05.

Results

Controls and treated birds were not significantly different in regard to body weight change over the study period (p=0.299). Renal histopathology revealed statistically significant changes (in renal tubule scores) in treated birds as compared to controls for the following microscopic lesions: PT degeneration (p=0.002), DT degeneration (p=0.002), DT inflammation (p=0.041), and intraluminal urate deposition (p=0.001) in the tubules. PT necrosis (p=0.115), PT inflammation (p=1.0), and DT necrosis (p =0.136) were not statistically different between the treatment and control groups. Inducing gentamicin toxicity caused no significant effect on body weight in treated as compared to control birds. Changes in levels of urine NAG (88.1 fold, p≤0.001) and creatine (5.7 fold; p≤0.001) from baseline were both significantly elevated in treated birds, as compared to controls. Changes in plasma NAG (6.3 fold; p=0.019) and uric acid (54.9 fold; p=0.033) were significantly elevated in treated as compared to control birds, whereas, creatine while elevated, was not significantly so, apparently, due to large variances (22.5 fold; p=0.387).

Discussion

Proximal tubular damage is commonly reported in a range of species in response to gentamicin toxicity. Distal tubular damage observed could reflect the dose, secondary hypoxia, or some as yet unexplained effect of gentamicin. Of the three markers evaluated in plasma, NAG and uric acid were elevated, but the former exhibited less variability in response to gentamicin exposure. In urine, creatine and NAG were significantly elevated, while uric acid is typically precipitated and is not reliably measured. The observation that creatine was elevated in the urine but not in the plasma needs to be more closely evaluated. This observation may suggest rapid renal clearance, a small volume of distribution, and/or variation in the time course of creatine released from muscle in response to gentamicin injections. For example, assessing the degree of muscle necrosis, (e.g., CPK) may allow creatine elevations to be better interpreted during renal disease assessment.

Although preliminary, these studies suggest the potential to noninvasively screen urine samples for renal dysfunction in the future, and hopefully will allow clinicians to better detect disease and prevent serious renal disease outcomes. The potential to identify acute disease, and to sample either blood or urine to identify a nascent renal toxic insult as reported here, if supported by further studies in a range of species, could improve the outcomes for captive avians with renal disease in the future. These noninvasive methods might also be adapted for use during wild bird health surveys; for example, to assess the impact of lead at wildlife refuges, and in response to other nephrotoxic exposures in the wild.

Acknowledgments

The authors would like to thank Dr. M. Fettman for several helpful suggestions.

Literature Cited

1.  Styles, DK, and DN Phalen. 1998. Clinical Avian urology. Semin Av Exot Pet Med. 7(2):104–113.

2.  Wimsatt J, RD Pearce, S Nelson, LT Shanahan and LM Vap. 2003. Tissue content of novel renal disease markers in pigeons (Columba livia). AAZV proceedings, Oct. 4–10. Minneapolis, MN, p. 317–318.