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A Drosophila model identifies a critical role for zinc in mineralization for kidney stone disease.

Chi T, Kim MS, Lang S, Bose N, Kahn A, Flechner L, Blaschko SD, Zee T, Muteliefu G, Bond N, Kolipinski M, Fakra SC, Mandel N, Miller J, Ramanathan A, Killilea DW, Brückner K, Kapahi P, Stoller ML - PLoS ONE (2015)

Bottom Line: To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation.Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation.Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Ectopic calcification is a driving force for a variety of diseases, including kidney stones and atherosclerosis, but initiating factors remain largely unknown. Given its importance in seemingly divergent disease processes, identifying fundamental principal actors for ectopic calcification may have broad translational significance. Here we establish a Drosophila melanogaster model for ectopic calcification by inhibiting xanthine dehydrogenase whose deficiency leads to kidney stones in humans and dogs. Micro X-ray absorption near edge spectroscopy (μXANES) synchrotron analyses revealed high enrichment of zinc in the Drosophila equivalent of kidney stones, which was also observed in human kidney stones and Randall's plaques (early calcifications seen in human kidneys thought to be the precursor for renal stones). To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation. Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation. Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.

No MeSH data available.


Related in: MedlinePlus

Inhibition of xanthine dehydrogenase leads to ectopic calcification in the fly Malpighian tubules.(A) Representative tubule images taken from control flies (Da-GAL4/+, left panel) and flies upon Xdh knockdown (Da-GAL4, UAS-Xdh RNAi /+, right panel). Concretions are dark and intraluminal. Scale bars: 500 μm. (B) HPLC-MS (multiple reaction monitoring) analysis of whole flies demonstrates significant reduction in uric acid levels in Da-GAL4, UAS-Xdh RNAi /+ flies compared to control flies with concomitant increase in xanthine and hypoxanthine levels. (C) Confocal microscopy of a fly tubule concretion isolated after Xdh knockdown. The gross appearance resembles that of a miniaturized kidney stone. Scale bar: 20 μm. (D) HPLC-MS (multiple reaction monitoring) analysis of concretions taken from these Da-GAL4, UAS-Xdh RNAi /+ flies demonstrates that they primarily contain xanthine and hypoxanthine with smaller amounts of uric acid and other purine metabolites also present. (E) μXANES demonstrates the presence of hydroxyapatite as the primary calcium salt in Drosophila concretion samples.
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pone.0124150.g001: Inhibition of xanthine dehydrogenase leads to ectopic calcification in the fly Malpighian tubules.(A) Representative tubule images taken from control flies (Da-GAL4/+, left panel) and flies upon Xdh knockdown (Da-GAL4, UAS-Xdh RNAi /+, right panel). Concretions are dark and intraluminal. Scale bars: 500 μm. (B) HPLC-MS (multiple reaction monitoring) analysis of whole flies demonstrates significant reduction in uric acid levels in Da-GAL4, UAS-Xdh RNAi /+ flies compared to control flies with concomitant increase in xanthine and hypoxanthine levels. (C) Confocal microscopy of a fly tubule concretion isolated after Xdh knockdown. The gross appearance resembles that of a miniaturized kidney stone. Scale bar: 20 μm. (D) HPLC-MS (multiple reaction monitoring) analysis of concretions taken from these Da-GAL4, UAS-Xdh RNAi /+ flies demonstrates that they primarily contain xanthine and hypoxanthine with smaller amounts of uric acid and other purine metabolites also present. (E) μXANES demonstrates the presence of hydroxyapatite as the primary calcium salt in Drosophila concretion samples.

Mentions: Seeking a Drosophila model for urinary stone disease, we examined the consequences of knocking down orthologs of human genes implicated in kidney stone formation on mineralized concretion formation in adult Drosophila Malpighian tubules. From a screen of ten such genes we observed a strong incidence of concretion formation upon inhibition of xanthine dehydrogenase (Xdh). Human mutations in xanthine dehydrogenase activity result in the disorder xanthinuria type I. An equivalent disease also occurs in dogs, most notably the Cavalier King Charles spaniel breed [18]. Patients with xanthinuria type I, one of the few single gene disorders known to result in nephrolithiasis in humans, form debilitating, recurrent kidney stones [19]. We observed that RNAi inhibition of Xdh [20] resulted in significantly increased tubule concretion formation when compared to controls (Fig 1A). RNAi knockdown efficiency was confirmed with RT-PCR (S1 Fig). Under light microscopy examination, concretions were visible as dark intraluminal contents within the Malpighian tubule and had the appearance of small stones. Upon dissection they also looked like small stones and their hardness could be felt between ones’ fingers. Given their nature, we refer to these exuberant concretions as fly stones. To confirm that this fly stone accumulation phenotype was specific to the gene function and not due to strain background, stone presence was confirmed in a second RNAi line against Xdh (S2 Fig). Next, using high performance liquid chromatography (HPLC)—mass spectrometry (MS) (multiple reaction monitoring, MRM)-based targeted metabolomics for quantification of purines, we found that RNAi knockdown of Xdh indeed increased xanthine and hypoxanthine, and decreased uric acid (Fig 1B) levels in the whole fly, supporting the proposed biochemical role of Xdh. Furthermore, we also observed that allopurinol, a pharmacologic inhibitor of xanthine dehydrogenase activity, led to stone formation (S3 Fig). Thus, both genetic and pharmacological inhibition of xanthine dehydrogenase led to stone formation in D. melanogaster.


A Drosophila model identifies a critical role for zinc in mineralization for kidney stone disease.

Chi T, Kim MS, Lang S, Bose N, Kahn A, Flechner L, Blaschko SD, Zee T, Muteliefu G, Bond N, Kolipinski M, Fakra SC, Mandel N, Miller J, Ramanathan A, Killilea DW, Brückner K, Kapahi P, Stoller ML - PLoS ONE (2015)

Inhibition of xanthine dehydrogenase leads to ectopic calcification in the fly Malpighian tubules.(A) Representative tubule images taken from control flies (Da-GAL4/+, left panel) and flies upon Xdh knockdown (Da-GAL4, UAS-Xdh RNAi /+, right panel). Concretions are dark and intraluminal. Scale bars: 500 μm. (B) HPLC-MS (multiple reaction monitoring) analysis of whole flies demonstrates significant reduction in uric acid levels in Da-GAL4, UAS-Xdh RNAi /+ flies compared to control flies with concomitant increase in xanthine and hypoxanthine levels. (C) Confocal microscopy of a fly tubule concretion isolated after Xdh knockdown. The gross appearance resembles that of a miniaturized kidney stone. Scale bar: 20 μm. (D) HPLC-MS (multiple reaction monitoring) analysis of concretions taken from these Da-GAL4, UAS-Xdh RNAi /+ flies demonstrates that they primarily contain xanthine and hypoxanthine with smaller amounts of uric acid and other purine metabolites also present. (E) μXANES demonstrates the presence of hydroxyapatite as the primary calcium salt in Drosophila concretion samples.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4430225&req=5

pone.0124150.g001: Inhibition of xanthine dehydrogenase leads to ectopic calcification in the fly Malpighian tubules.(A) Representative tubule images taken from control flies (Da-GAL4/+, left panel) and flies upon Xdh knockdown (Da-GAL4, UAS-Xdh RNAi /+, right panel). Concretions are dark and intraluminal. Scale bars: 500 μm. (B) HPLC-MS (multiple reaction monitoring) analysis of whole flies demonstrates significant reduction in uric acid levels in Da-GAL4, UAS-Xdh RNAi /+ flies compared to control flies with concomitant increase in xanthine and hypoxanthine levels. (C) Confocal microscopy of a fly tubule concretion isolated after Xdh knockdown. The gross appearance resembles that of a miniaturized kidney stone. Scale bar: 20 μm. (D) HPLC-MS (multiple reaction monitoring) analysis of concretions taken from these Da-GAL4, UAS-Xdh RNAi /+ flies demonstrates that they primarily contain xanthine and hypoxanthine with smaller amounts of uric acid and other purine metabolites also present. (E) μXANES demonstrates the presence of hydroxyapatite as the primary calcium salt in Drosophila concretion samples.
Mentions: Seeking a Drosophila model for urinary stone disease, we examined the consequences of knocking down orthologs of human genes implicated in kidney stone formation on mineralized concretion formation in adult Drosophila Malpighian tubules. From a screen of ten such genes we observed a strong incidence of concretion formation upon inhibition of xanthine dehydrogenase (Xdh). Human mutations in xanthine dehydrogenase activity result in the disorder xanthinuria type I. An equivalent disease also occurs in dogs, most notably the Cavalier King Charles spaniel breed [18]. Patients with xanthinuria type I, one of the few single gene disorders known to result in nephrolithiasis in humans, form debilitating, recurrent kidney stones [19]. We observed that RNAi inhibition of Xdh [20] resulted in significantly increased tubule concretion formation when compared to controls (Fig 1A). RNAi knockdown efficiency was confirmed with RT-PCR (S1 Fig). Under light microscopy examination, concretions were visible as dark intraluminal contents within the Malpighian tubule and had the appearance of small stones. Upon dissection they also looked like small stones and their hardness could be felt between ones’ fingers. Given their nature, we refer to these exuberant concretions as fly stones. To confirm that this fly stone accumulation phenotype was specific to the gene function and not due to strain background, stone presence was confirmed in a second RNAi line against Xdh (S2 Fig). Next, using high performance liquid chromatography (HPLC)—mass spectrometry (MS) (multiple reaction monitoring, MRM)-based targeted metabolomics for quantification of purines, we found that RNAi knockdown of Xdh indeed increased xanthine and hypoxanthine, and decreased uric acid (Fig 1B) levels in the whole fly, supporting the proposed biochemical role of Xdh. Furthermore, we also observed that allopurinol, a pharmacologic inhibitor of xanthine dehydrogenase activity, led to stone formation (S3 Fig). Thus, both genetic and pharmacological inhibition of xanthine dehydrogenase led to stone formation in D. melanogaster.

Bottom Line: To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation.Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation.Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Ectopic calcification is a driving force for a variety of diseases, including kidney stones and atherosclerosis, but initiating factors remain largely unknown. Given its importance in seemingly divergent disease processes, identifying fundamental principal actors for ectopic calcification may have broad translational significance. Here we establish a Drosophila melanogaster model for ectopic calcification by inhibiting xanthine dehydrogenase whose deficiency leads to kidney stones in humans and dogs. Micro X-ray absorption near edge spectroscopy (μXANES) synchrotron analyses revealed high enrichment of zinc in the Drosophila equivalent of kidney stones, which was also observed in human kidney stones and Randall's plaques (early calcifications seen in human kidneys thought to be the precursor for renal stones). To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation. Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation. Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.

No MeSH data available.


Related in: MedlinePlus