The renal phenotype of allopurinol-treated HPRT-deficient mouse.

Cristina Zennaro,Paola Zarattini,Marialaura Marchetti,Gianluca Paredi,Federica Tonon,Alessandro Corbelli,Milan Clai,Michele Carraro,Maria Pia Rastaldi,Luca Ronda,Riccardo Percudani

doi:10.1371/journal.pone.0173512

Abstract

Excess of uric acid is mainly treated with xanthine oxidase (XO) inhibitors, also called uricostatics because they block the conversion of hypoxanthine and xanthine into urate. Normally, accumulation of upstream metabolites is prevented by the hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme. The recycling pathway, however, is impaired in the presence of HPRT deficiency, as observed in Lesch-Nyhan disease. To gain insights into the consequences of purine accumulation with HPRT deficiency, we investigated the effects of the XO inhibitor allopurinol in Hprt-lacking (HPRT-/-) mice. Allopurinol was administered in the drinking water of E12-E14 pregnant mothers at dosages of 150 or 75 μg/ml, and mice sacrificed after weaning. The drug was well tolerated by wild-type animals and heterozygous HPRT+/- mice. Instead, a profound alteration of the renal function was observed in the HPRT-/- model. Increased hypoxanthine and xanthine concentrations were found in the blood. The kidneys showed a yellowish appearance, diffuse interstitial nephritis, with dilated tubules, inflammatory and fibrotic changes of the interstitium. There were numerous xanthine tubular crystals, as determined by HPLC analysis. Oil red O staining demonstrated lipid accumulation in the same location of xanthine deposits. mRNA analysis showed increased expression of adipogenesis-related molecules as well as profibrotic and proinflammatory pathways. Immunostaining showed numerous monocyte-macrophages and overexpression of alpha-smooth muscle actin in the tubulointerstitium. In vitro, addition of xanthine to tubular cells caused diffuse oil red O positivity and modification of the cell phenotype, with loss of epithelial features and appearance of mesenchymal characteristics, similarly to what was observed in vivo. Our results indicate that in the absence of HPRT, blockade of XO by allopurinol causes rapidly developing renal failure due to xanthine deposition within the mouse kidney. Xanthine seems to be directly involved in promoting lipid accumulation and subsequent phenotype changes of tubular cells, with activation of inflammation and fibrosis.

Highlights

Lesch-Nyhan disease (LND) is a rare X-linked monogenic disorder caused by an inborn error of nucleotide metabolism
A direct consequence of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency is the augmented degradation of free purines and results in overproduction of the end-product uric acid, which accumulates in plasma and urine
Treatment with allopurinol had highly toxic effects in HPRT-/- mice, as compared to the same drug dosages administered to HPRT+/- and wild type (WT) animals

Summary

Introduction

Lesch-Nyhan disease (LND) is a rare X-linked monogenic disorder caused by an inborn error of nucleotide metabolism. The mutations are localized on the gene HPRT1, which encodes the enzyme hypoxanthine-guanine phosphoribosyltransferase, a key molecule in recycling purine bases. A direct consequence of HPRT deficiency is the augmented degradation of free purines and results in overproduction of the end-product uric acid, which accumulates in plasma (hyperuricemia) and urine (hyperuricosuria). Hyperuricosuria leads to formation of sandy sludge or stones in the urinary collecting system, causing renal failure if not promptly treated [1]. The drug blocks the enzyme xanthine oxidase (XDH), thereby increasing the upstream formation of oxypurines, i.e. xanthine and hypoxanthine, which can be harmful to cell metabolism [2]

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Conclusion