183
Toxicology Letters, 53 (1990) 183-185
Elsevier TOXLET 02412
A quantitative method for the measurement of urinary glycosaminoglycans - potential use in studies of xenobiotic nephrotoxicity S. Halligan, M.J. Graham, T.J.B. Gray, E.S. Harpur and F.W. Bonner Sterling Research Group, Alnwick, Northumberland
(U.K.)
Key words: Renal papillary necrosis; Glycosaminoglycans;
2-Bromoethanamine
hydrobromide
Although renal papillary necrosis (RPN) is a common drug-associated nephropathy [l], the molecular mechanisms involved have not yet been fully elucidated and there are no established biochemical indices of the lesion. Our interest lies in the development of sensitive and specific early markers of RPN for use both in clinical diagnosis and as a screen during drug development. Since the renal papilla is rich in glycosaminoglycans (GAGS) [2], we have investigated the potential use of changes in urinary GAG excretion as an early non-invasive marker of xenobiotic-induced RPN. The rationale for such an approach originates from the observation that some of the earliest changes reported in experimental models of RPN involve degeneration of interstitial cells and the interstitial matrix [3] and that autopsy material from rats treated with the model renal papillary toxin 2bromoethanamine hydrobromide (BEA) have shown changes in the histochemical staining of GAGS [l], indicating a change in GAG content or structure. The two analytical techniques most commonly employed for the quantitative measurement of urinary GAGS are the alcian blue dye-binding method [4] and the carbazole borate assay, which involves the reaction of carbazole with the dehydrated derivatives of uranic acid resulting from the acid hydrolysis of GAGS [5]. The alcian blue technique has the advantage of speed and simplicity; however, in our hands it proved to be unsatisfactory for use with rat urine due to deviations from linearity and poor reproducibility. In contrast, the carbazole-borate method was both linear and reproducible up to at least 600 pg/ml of chondroitin sulphate, a typical GAG. Address for correspondence:
S. Halligan, Sterling Research Group, Alnwick, Northumlxrland
U.K. 0378-4274/90/$3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
NE66 2JH,
184
Since urine contains large quantities of free uranic acid and other low-molecularweight interfering substances, extraction of GAGS is necessary prior to analysis. Extraction is most commonly achieved by precipitation with quaternary ammonium compounds. However, the initial isolation of GAGS from rat urine by precipitation with cetylpyridinium chloride was found to be incomplete and poorly reproducible. Consequently, the gel filtration method for urinary GAG isolation described by Hurst et al. [6] was adopted, with a number of modifications. The separations (sample volume 5 ml) were performed on columns (1.5 x 50 cm) packed with Biogel P-4, 100-200 mesh (BioRad Laboratories) to a bed height of 12 cm. Columns were eluted with 0.01 M NaCl delivered at a constant flow rate of 1.2 ml/min. The gel-filtration elution profile of rat urine indicated the presence of two peaks. The first was eluted in the void volume and comprised GAGS and high-molecularweight proteins while the second fraction contained low-molecular-weight proteins, free uranic acid and other interfering substances. Thus, the macromolecular fraction was collected, lyophilised and reconstituted in distilled water prior to analysis by the carbazole-borate method. This method provided complete and rapid separation of GAGS from other urinary carbazole-borate-reactive material. Using this procedure, changes in urinary GAG excretion were measured during investigations of xenobiotic-induced RPN. An initial time-course study indicated that the administration of a single intraperitoneal dose of BEA (150 mg/kg) to female Sprague-Dawley rats (n=5), resulted in a 2.5-fold increase in group mean urinary GAG excretion during the period O-8 h post-dose, compared to saline-treated controls (n = 5). Urinary GAG excretion remained elevated (1.5-fold) during the period 8-24 h post-dose before returning to control levels between 24 and 48 h post-dose. Based on these results, a further study was conducted using female Sprague-Dawley rats, housed individually in metabolism cages and administered a single intraperitoneal injection of either 150 mg/kg BEA (n= 10) or 0.9% saline (n =4). Urine was collected (324 h pre- and post-dose and total GAGS excreted in the urine over each collection period were determined and expressed as mg uranic acid excreted per mmol creatinine. The total urinary GAG excretion (mean + SD) of the BEA-treated group increased significantly (P
Toxicology Letters, 53 (1990) 183-185
Elsevier TOXLET 02412
A quantitative method for the measurement of urinary glycosaminoglycans - potential use in studies of xenobiotic nephrotoxicity S. Halligan, M.J. Graham, T.J.B. Gray, E.S. Harpur and F.W. Bonner Sterling Research Group, Alnwick, Northumberland
(U.K.)
Key words: Renal papillary necrosis; Glycosaminoglycans;
2-Bromoethanamine
hydrobromide
Although renal papillary necrosis (RPN) is a common drug-associated nephropathy [l], the molecular mechanisms involved have not yet been fully elucidated and there are no established biochemical indices of the lesion. Our interest lies in the development of sensitive and specific early markers of RPN for use both in clinical diagnosis and as a screen during drug development. Since the renal papilla is rich in glycosaminoglycans (GAGS) [2], we have investigated the potential use of changes in urinary GAG excretion as an early non-invasive marker of xenobiotic-induced RPN. The rationale for such an approach originates from the observation that some of the earliest changes reported in experimental models of RPN involve degeneration of interstitial cells and the interstitial matrix [3] and that autopsy material from rats treated with the model renal papillary toxin 2bromoethanamine hydrobromide (BEA) have shown changes in the histochemical staining of GAGS [l], indicating a change in GAG content or structure. The two analytical techniques most commonly employed for the quantitative measurement of urinary GAGS are the alcian blue dye-binding method [4] and the carbazole borate assay, which involves the reaction of carbazole with the dehydrated derivatives of uranic acid resulting from the acid hydrolysis of GAGS [5]. The alcian blue technique has the advantage of speed and simplicity; however, in our hands it proved to be unsatisfactory for use with rat urine due to deviations from linearity and poor reproducibility. In contrast, the carbazole-borate method was both linear and reproducible up to at least 600 pg/ml of chondroitin sulphate, a typical GAG. Address for correspondence:
S. Halligan, Sterling Research Group, Alnwick, Northumlxrland
U.K. 0378-4274/90/$3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
NE66 2JH,
184
Since urine contains large quantities of free uranic acid and other low-molecularweight interfering substances, extraction of GAGS is necessary prior to analysis. Extraction is most commonly achieved by precipitation with quaternary ammonium compounds. However, the initial isolation of GAGS from rat urine by precipitation with cetylpyridinium chloride was found to be incomplete and poorly reproducible. Consequently, the gel filtration method for urinary GAG isolation described by Hurst et al. [6] was adopted, with a number of modifications. The separations (sample volume 5 ml) were performed on columns (1.5 x 50 cm) packed with Biogel P-4, 100-200 mesh (BioRad Laboratories) to a bed height of 12 cm. Columns were eluted with 0.01 M NaCl delivered at a constant flow rate of 1.2 ml/min. The gel-filtration elution profile of rat urine indicated the presence of two peaks. The first was eluted in the void volume and comprised GAGS and high-molecularweight proteins while the second fraction contained low-molecular-weight proteins, free uranic acid and other interfering substances. Thus, the macromolecular fraction was collected, lyophilised and reconstituted in distilled water prior to analysis by the carbazole-borate method. This method provided complete and rapid separation of GAGS from other urinary carbazole-borate-reactive material. Using this procedure, changes in urinary GAG excretion were measured during investigations of xenobiotic-induced RPN. An initial time-course study indicated that the administration of a single intraperitoneal dose of BEA (150 mg/kg) to female Sprague-Dawley rats (n=5), resulted in a 2.5-fold increase in group mean urinary GAG excretion during the period O-8 h post-dose, compared to saline-treated controls (n = 5). Urinary GAG excretion remained elevated (1.5-fold) during the period 8-24 h post-dose before returning to control levels between 24 and 48 h post-dose. Based on these results, a further study was conducted using female Sprague-Dawley rats, housed individually in metabolism cages and administered a single intraperitoneal injection of either 150 mg/kg BEA (n= 10) or 0.9% saline (n =4). Urine was collected (324 h pre- and post-dose and total GAGS excreted in the urine over each collection period were determined and expressed as mg uranic acid excreted per mmol creatinine. The total urinary GAG excretion (mean + SD) of the BEA-treated group increased significantly (P
