Pathophysiology of diclofenac-associated hyperuricaemia in Gyps spp. vultures

Abstract

Diclofenac toxicity in old world vultures is well described in the literature. As part of the toxicity of the drug, dead birds were generally found in the environment with signs of severe renal damage and gout. In birds that were tested before death, signs of hyperuricaemia and hyperkalaemia were also present. Although the clinical picture is very clear, the sequence of hyperuricaemia is not yet established with one possibility being drug induced renal damage leading to hyperuriceamia and the second being secondary kidney damage resulting from hyperuricaemic cellular damage. For this study we evaluated this sequence by assessing the effect of diclofenac on uric acid transporters in the chicken, a validated model for diclofenac toxicity. We speculated that diclofenac, a known uricosuric drug in people, inhibits the avian organic anionic transporters (OATs) with subsequent increase in plasma uric acid, precipitation and kidney damage. As a first step, the impact of diclofenac was evaluated in healthy chicken as it was not justifiable to kill vultures through diclofenac administration. For this two-phase study heathy chickens were treated intravenous with para-amino hippuric acid (PAH) and iohexol (IOH) in combination in phase 1 or this same combination with diclofenac (10 mg/kg) in phase 2. In both phases blood and faeces were sequentially collected. In phase 1, birds showed no signs of ill health, moreover PAH, IOH and uric acid clearance was rapid. In phase 2, two chickens eventually died with hyperuricaemia being present as soon as 8 hours after exposure. Necropsy showed classic signs of renal damage and hyperuricaemia. Pharmacokinetic analysis revealed a rapid half-life of elimination of less than 2 hours indicating that toxicity was due to irreversible inhibition of a physiological process. In phase 2 all the birds had decrease uric acid, PAH and IOH clearance. While tubular excretory rates of PAH were reduced in all birds in phase 2, they were 98% reduced in the two birds that died. Based on the global change in clearance parameters, it is concluded that diclofenac alters both renal perfusion (IOH measures glomerular filtration) and renal plasma flow. However death results from tubular secretion being reduced to negligible functionality for a prolonged period. With birds being highly reliant on tubular secretion of uric acid, we conclude that diclofenac hyperuricaemia is as a result of OAT inhibition and that cell death results secondarily from uric acid precipitation as described in gouty chicken. In the final conclusive study, we used next generation sequencing of the transcriptome of the renal tissue from one African white backed vulture (AWB), to establish if these tissues expressed OATs and multidrug resistance protein (MRP) transporters. Both these channels are known to be involved with uric acid transport either basolaterially or apically respectively. The Trinity assembled transcriptome, was used to create a local blast database from which predicted sequences of OAT1 and 2 and MRP2 and 4 from the Golden Eagle were evaluated for similarity. The golden eagle was selected as it was the closest related species phylogenetically that also had a complete genome sequence published. OAT3 was not included in this study as no avian sequence was available on the NCBI database. From the search, all four channels were identified in the AWB vulture kidney with high similarity to the golden eagle. Sanger sequencing confirmed the presence of the OAT 1, 2 and MRP2, 4 related mRNA. The predictive amino acid sequence and predictive protein channel (Swiss-Prot) was also used to provide some evidence that the proteins in question shared the required characteristic and function of OAT and MRP channels. After in silico analysis revealed the similarity of only AWB OAT2 gene with other species i.e chicken, expression study was carried out. It revealed that chicken OAT2 gene was expressed more than the vulture, this maybe the reason to vulture sensitivity to diclofenac. With the genes showing the presence of the requisite uric acid transport proteins in the kidney, the distribution of the OAT channels in the vulture was confirmed by immunohistochemistry (IHC) using mouse polyclonal OAT3 antibodies as sequence analysis showed high similarity between vulture OAT1 and mouse OAT3. As expected the IHC showed the presence of OAT1 with good distribution along the renal tubules.