CROSS REFERENCE ^' TO RELATED APPLICATIONS

This application claims the prioriiy benefit of U.S. Provisional Patent Application Serial No. 61/648,929 filed May 18, 2012, and U.S. Provisional Patent Application Serial No.

61/660,913 filed June 18, 2012.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Contract Nos. NIH R01

AG031337-01A 1, ROl AG038688, R i . i AAG0321 13, P01 AG025901-S 1, and K12-DK-07-006 awarded by the National Institutes of Health, The U.S. Government as certain rights in this invention.

BACKGROUND OF THE INVENTION

1) FIELD OF THE INVENTION

The field of the present invention is the treatment of urinary tract sto es.

2) DESCRIPTION OF RELATED ART

Nephrolithiasis accounted for more than S2.1 billion in medical expenditures in the United States in 2000, encompassing costs in both the outpatient and inpatient settings [Pearie, M. et ai. (2005) J. Urol. 173, 848-857], Symptomatic kidney stones are a source of severe pain, infection, and morbidity. Consequently, new insights that would lead to effective medical treatment of stone formation would reduce the morbidity and costs associated with this significant disease. Current animal models of nephrolithiasis have not led to tangible progress in understanding the genetics and mechanisms behind stone formation or development of new medical treatments for nephrolithiasis. Though several models have been developed, none are without significant limitations. For example, mice fed with glyoxylic acid and pigs fed with hydroxyproline are used to generate calcium oxalate stones [Kapion, D. M. et ai. (2010) J. Endouroi. 2.4, 355—359] . However, neither glyoxylic acid nor hydroxyproline are normal dietary components for animals or humans to consume in excess and, as such, these models have questionable Iranslational significance. While much progress has been made in surgical therapy for kidney stones, no new FDA-approved drag for prevention of nephrolithiasis has been developed in more than 20 years.

Additionally, it has been shown that patients with nephrolithiasis (kidney stone disease) have an increased likelihood of developing cardiovascular disease, including hypertension, myocardial infarction, congestive heart failure, and coronary artery calcification [Reiner, A. P. et al. (201 1) J. Urol. 185, 920-925]. The mineralization process underlying early stone formation may be a common linkage between these systemic disorders. Bio mineralization also plays a pivotal role in a number of pathologic conditions as diverse as atherosclerosis [Dow, J. T. & Davies, S. A. (2003) Physiol. Rev. 83, 687-729] and neurodegenerative disorders [Dow, J. A. T. & Davies, S. A. (2006) J. Insect. Physiol. 52, 365-378]. Biomineralization typically involves calcification, for which several factors have been implicated. Calcium hydroxyapatite has been thought to serve as a nidus for the formation of a variety of mineralized deposits and structures [Pearle, M. et al. (2005) J. Urol. 173, 848-857; Arikyants, N. et al. (2007) Pediafx Nephrol 22, 310-314; Maalouf, N. M. et al. (201 1 ) J. Clin. Endocrin. and Metab. 96, 3733-3740], but the driving forces initiating the calcification process are not well understood.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a table showing Drosophila melanogaster genes that were screened for increased concretion formation. UAS-RNAi lines for each gene of interest were crossed with GAL4-Da virgins and progeny were examined on protein, salt, and calcium supplementation for the presence of increased concretions compared to wild type controls.

Fig. 2 (A & B) shows that a high protein diet increases fly stone formation upon XDH knockdown. (A) Male XDH knockdown (+/Da-GAL4; +/UAS-XDH RNAi) flies were reared on a normal diet (1 .5% YE) on day 1 (left panel), and then transferred to either rich diet (5% yeast) (middle panel) or dietary restriction condition (0.5% yeast) (right panel). Two days later (day 3), the level of stone formation in each group was quantified. (B) Quantification of the stone formation in XDH knockdown animals under different dietary manipulations. Flies with XDH knockdown (black bars) fed on a high protein diet containing 5% YE (5% protein) compared to a low protein diet containing 0.5% YE (0.5% protein) develop statistically significant greater stone formation (p<0.001 ).

Fig. 3 (A - D) shows that Drosophila produce stones, a form of biomineralization similar to human kidney stones. (A) Dissected Malpighian tubules from Da-GAL4, UAS-Xdh RNAi /+ male flies demonstrate stones occupying the tubule lumen, as indicated by the arrow (left panel). Alexa-488 fluorescent dye bound to alendronate demonstrates strong affinity for calcium salts in these stones (right panel). (B) μΧΑΝΕ8 demonstrates the presence of hydroxyapatite as the primary calcium salt in Drosophila stones as well as human Randall's plaque samples. (C) X-ray diffraction pattern of Drosophila stones utilizing a micro foe used synchrotron beam of 14 keV energy demonstrates that stones are crystalline in nature. (D) ICP- OES analysis of pooled fly stone samples from 300 dissected tubule specimens demonstrates the presence of calcium, magnesium, and zinc (n = 2 biological replicates). Data shown are the mean + SEM.

Fig. 4 (A - D) shows that Drosophila concretions resemble human Randall plaques and have a similar chemical composition. (A) Gross photomicrographs of concretions isolated from the Malpighian tubules of male Da-GAL4, UAS-XDH RNAi /+ flies fed on a 5% yeast diet (upper panel) and a human renal papilla biopsy (lower panel). The dark areas of the biopsy- sample (lower panel) represent Randall's plaque material. (B) Transmission electron microscopy imaging of concretions in the lumen of the Malpighian tubule demonstrates the presence of ringlike structures, as indicated by the yellow arrows (upper panel). Ring structures with homologous appearance are seen in Randall's plaques taken from a human renal papilla biopsy material (lower panel) when imaged in a similar fashion. (C) Micro-X-ray fluorescence QiXRF) maps of Da-GAL4, UAS-XDH RNAi /+ concretions (upper panel) and human Randall's plaques (lower panel) demonstrate the presence of zinc in red and calcium in green. (D) μΧΚΡ elemental analysis of the samples from (C) demonstrate similar elemental composition for both Da-GAL4, UAS-XDH RNAi /+ concretions (upper panel) and human Randall's plaques (lower panel), including the presence of calcium (Ca), iron (Fe), and zinc (Zn).

Fig. 5 (A-D) shows that zinc modulates stone formation in fly tubules. (A) Da-Gal4, UAS-Xdh RNAi/+ male flies fed on 0.5% YE and 5% YE diets were supplemented with different doses of Zn. This led to a dose-dependent increase in accumulation of stones on 0.5% YE feeding (*p<0.05, ***p<0.001 , student t-test, n= 10-48). Zn supplementation had no effect on altering stone formation in 5% YE-fed animals, which may reflect a saturation of the mineralization process under these conditions. (B) Supplementation of 5% YE diet with the zinc chelator TPEN reduced stone formation. This effect could be reversed with the addition of 10 niM Zn but not 10 ml Mg (*p<0.05, ***p<0.Gl, one way ANOVA, n=10-43). (C) On 5% YE, simultaneous inhibition of xanthine dehydrogenase (Da~GAL4, UAS-Xdh RNAi) and three different zinc transporters (UAS-CG1 1 163 RNAi, UAS-CG17723 RNAi, and UAS-CG3994 RNAi) resulied in statistically significant decreased stone accumulation compared to DaGAL4, UAS-Xdh RNAi/+ flies (***p<0.001 , one way ANOVA, n=14-43). (D) Simultaneous inhibition of the same three zinc transporters with Xdh rescued survivorship when compared with Da- GAL4, UAS-Xdh RNAi/+ animals on 5% YE (logrank test, n=76-97). Data shown are the mean + SEM or mean - SEM,

DETAILED DESCRIPTION OF THE INVENTION

Provided herein is a method of reducing the formation of a urinary tract stone in a subject in need of such reduction. It is a surprising finding of the present invention that urinary tract stones can be reduced in a subject by reducing the bioavailability of zinc ions in the subject. Accordingly, the present invention provides a ne method of treatment for those subjects that are prone to the development of urinary tract stones or those subjects that wish to avoid the development of urinary tract stones. Term definitions used in the specification and claims are as follows.

Definitions

As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a ceil" includes a plurality of cells, including mixtures thereof.

When referring to a subject or patient, the term "administering" refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, imramuscular, intra-joint, parenteral, intra- arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term "parenteral" includes subcutaneous, intravenous, intramuscular, intraarticular, intra-peritoneal, intra-synovial, intraster af, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. In some embodiments, the administration is intranasal. In other embodiments, the administration is intravenous. As also used herein, the term "systemic administration" refers to an administration that requires the administered composition to cross the blood-brain barrier in order to reach the brain.

As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.

An "effective amount" is an amount sufficient to effect beneficial or desired results. For example, in some embodiments, an effective amount of a composition reduces urinary tract sto es in a subject to whom it is administered. An effective amount can be administered in one or more administrations, applications, or dosages.

As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell, "Overexpression" as applied to a gene refers to the overproduction of the mRNA transcribed from the gene or the protein product encoded by the gene at a level that is 2.5 times higher, preferably 5 times higher, more preferably 10 times higher, than the expression level detected in a control sample. "Reducing expression" as applied to a gene refers to the underproduction of the mRNA transcribed from the gene or the protein product encoded by the gene at a level that is 2.5 times lower, preferably 5 times lower, more preferably 10 times lower, than the expression level detected in a control sample.

A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art, some of which are described herein. A "gene product" refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.

The term "isolated" means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof are normally associated with in nature. In one aspect of this invention, an isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non- naturally occurring polynucleotide, peptide, polypeptide, protein, or antibody, or fragments thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart. In addition, a "concentrated," "separated," or "diluted" polynucleotide, peptide, polypeptide, protein, or antibody, or fragments thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than "concentrated" or less than "separated" than that of its naturally occurring counterpart. A polynucleotide, peptide, polypeptide, protein, or antibody, or fragments thereof, which differs from the naturally occurring counterpart in its primary sequence or for example, by its glycosylation pattern, need not be preseni in its isolated form since it is distmguishabie from its naturally occurring counterpart by its primary sequence, or alternatively, by another characteristic such as glycosylation pattern. Although not explicitly stated for each of the inventions disclosed herein, it is to be understood that all of the above embodiments for each of the compositions disclosed below and under the appropriate conditions are provided by this invention. Thus, a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide. A protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eukaryotic cell in which it is produced in nature.

The term "kidney tissue" includes, but is not limited to, nephron tissue, renal tubule tissue, calceys tissue, and kidney pelvis tissue. Each human kidney contains approximately one million nephrons. These nephrons are the location where the urinary tract system and the blood system interact and materials are exchanged between the two systems. There are two types of nephrons: the cortical nephron and the juxtameduilary nephron. Kidney nephrons comprise a Bowmans capsule, proximal convoluted tubule, Loop of Henle, distal convoluted tubule, and collecting duct, in some embodiments, the kidney tissue is a convoluted tubule tissue such as a proximal convoluted tubule tissue and/or a distal convoluted tubule tissue. A "kidney stone" is a urinary tract stone produced or formed by a kidney tissue. A "pharmaceutical composition" is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

The term "pharmaceutically acceptable carrier or excipient" means a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically accepiable carrier or excipient" as used in the specification and claims includes both one and more than one such carrier or excipient. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservativ es.

The term "pharmaceutically acceptable salts" refers to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts. Specific examples of pharmaceutically acceptable salts are provided below.

The terms "pharmaceutically effective amount," "therapeutically effective amount," or "therapeutically effective dose" refer to the amount of a compound th t will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. In some embodiments, a pharmaceutically or therapeutically effective amount of a composition is the amount that reduces urinary tract stones in a subject to whom it is administered as compared to a control subject.

The terms "pharmaceutically effective amount" and "therapeutically effective amount" include that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent one or more of the symptoms of, the condition or disorder being treated. The therapeutically effective amount will vary depending on the compound, the disorder or conditions and their severity, the route of administration, time of administration, rate of excretion, drag combination, judgment of the treating physician, dosage form, and the age, weight, general health, sex and/or diet of the subject to be treated.

The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (niRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, RNAi, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, polynucleotide probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If preseni, modifications io the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double- stranded form.

A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine (T) when the polynucleotide is RNA. Thus, ihe term "polynucleotide sequence" is ihe alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching,

The term "polypeptide" is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. As used herein the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.

As used herein, the term "reducing transport of zinc ions" includes both reducing the uptake and the export of zinc ions from a cell. In some embodiments, the export of zinc ions from a cell is reduced. In other embodiments, the import of zinc ions into a cell is reduced. In still other embodiments, the export and import of zinc ions by a cell is reduced. It should be understood that "transport of zinc ions by a cell" refers to the movement of zinc ions across a cell 's membrane, or in other words, movement of zinc ions into or out of a cell. "Selectively binds" refers to a non-specific binding event as determined by an appropriate comparative control. Binding is selective when the binding is at least 10, 30, or 40 times greater than that of background binding in the comparative control.

As used herein, the term "stone forming bioavailability" refers to the ability of a zinc ion to form a urinary tract stone. A reduction in the stone forming bioavailability of a zinc ion can be achieved, for example, by the binding of the zinc ion to another substance such as a zinc chelator and/or by the reduced transport of zinc across a cell membrane.

A "subject" refers to a multicellular organism. The term "subject" includes vertebrates, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Subjects that are in need of reduced formation of urinary tract siones are those that are prone to developing urinary tract stones due to their genetic constituency, diet, or lifestyle. Some subjects prone to developing urinary tract stones are those who have low fluid intake, calcium supplements, and/or high dietary intake of animal protein, sodium, refined sugar, fructose, high fructose corn syrup, oxalate, grapefruit juice, apple juice, and/or cola drinks.

"Transformation" of a cellular organism with DNA means introducing DNA into an organism so that the DNA is repiicable, either as an extrachromosomal element or by chromosomal integration. "Transfection" of a cellular organism with DNA refers to the taking up of DNA, e.g., an expression vector, by the cell or organism whether or not any coding sequences are in fact expressed. The terms "transfected host cell" and "transformed" refer to a cell in which DNA was introduced. The cell is termed "host ceil" and it may be either prokaryotie or eukaryotic. Typical prokaryotie host cells include various strains of E. coii. Typical eukaryotic host cells are mammalian, such as Chinese hamster ovary or cells of human origin. The introduced DNA sequence may be from the same species as the host cell or from a different species than the host cell, or it may be a hybrid DNA sequence, containing some foreign and some homologous DNA.

As used herein, the term "urinary tract" includes a kidney, renal pelvis, ureter, bladder, and urethra.

A "urinary tract stone" is a stone located within urine in the urinary tract. Accordingly, urinary tract stones can be found and/or formed in the urinary tract. As used herein, the term "stone" (also referred to herein as a "concretion") refers herein to a relatively solid mass comprised of various compounds such as calcium, uric acid, struvite and/or cystine. Urinary tract stones include those comprised largely of calcium (calcium stone), uric acid (uric acid stone), struvite (stravite stone) or cystine (cystine stone). It should be understood that each type of stone can be comprised of numerous additional compounds in addition to calcium, uric acid, struvite, and cystine, respectively. In some embodiments, the stone comprises zinc as the additional compound. In some embodiments of the present method, the urinary tract stone is a calcium stone. In some further embodiments, the calcium stone comprises zinc. An "upper urinary tract stone" can form in the kidney, whereas a "lower urinary tract stone" can form in the ureter or bladder as a result of crystal formation in the urine.

As used herein, a "urinary tract tissue" includes a tissue of the kidney, renal pelvis, ureter, bladder, and urethra.

The term "vector" means a DMA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DMA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control the terminatson of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome or may, in some insiances, integrate into the genome itself. In the present specification, "plasmid" and "vector" are sometimes used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of vectors which serve equivalent functions as those in the art and which are, or become known, in the art.

As used herein, the term "zinc chelating agent" refers to a compound that binds a zinc ion and thereby prevents transport of the zinc ion across a cell membrane. In some embodiments, a zinc chelating agent prevents transport of a zinc ion across the membrane of a kidney tissue cell. In some further embodiments, a zinc chelating agents prevents transport of a zinc ion across the membrane of a kidney convoluted tubule cell. Non-limiting examples of zinc chelating agents are N,N,N',N' etra s"(2- yridylmethyi)ethylenedia:mme (TPEN), disulfiram, cloquinol, phytic acid, and thioflavins. As used herein, a "zinc transporter polypeptide" refers to a polypeptide involved in the transport of zinc ions across a cell membrane. Non-limiting examples of zinc transporter polypeptides include those polypeptides in the SLC30 (ZnT) family and SLC39 (ZIP) family of zinc transporters. ZnT proteins contribute to the cytoplasmic zinc balance by exporting zinc out to the extracellular space or by sequestering cytoplasmic zinc in intracellular compartments when cellular zinc levels are elevated. The ZnT family of genes currently comprises ien members SLC30A1 (ortliolog of D. melanogaster CGI 7723), SLC30A2 (ortholog of D. melanogaster CG3994), SLC30A3, SLC30A4 (ortholog of D. melanogaster CGI 1 163), SLC30A5, SLC30A6, SLC30A7, SLC30A8, SLC30A9, and SLC30A10. ZIP proteins function to increase cytoplasmic zinc concentrations when cellular zinc is depleted. The ZIP family of genes currently comprises ten members: SLC39A1, SLC39A2, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SLC39A7, SLC39A8, SLC39A10, and SLC39A14.

Discussion

Provided herein is a method of reducing the formation of a urinary tract stone in a subject in need of such reduction. It is a surprising finding of the present invention that urinary tract stones can be reduced in a subject by reducing the bioavailability of zinc ions in the subject. Accordingly, the present invention provides a new method of treatment for those subjects that are prone to the development of urinary tract stones or those subjects that wish to avoid the development of urinary tract stones.

The method provided herein comprises reducing stone forming bioavailability of zinc ions in a subject, thereby reducing formation of a urinary tract stone in the subject. As used herein, the term "stone forming bioavailability" refers to the ability of a zinc ion to form, initiate, or become a part of a urinary tract stone. A reduction in the stone forming bioavailability of a zinc ion can be achieved, for example, by the binding of the zinc ion to another substance such as a zinc chelator and/or by the reduced transport of zinc across a cell membrane. In some embodiments, the urinary tract stones are reduced in a subject treated by the methods described herein by approximately 95%, 80-90%, 70-75%, 50-60%, 25-30%, or 5- 10% as compared to a control subject,

A "urinary tract stone" is a stone located within urine in the urinary tract. Accordingly, urinary tract stones can be found and/or formed in the urinary tract. As used herein, the term "stone" refers herein to a relatively solid mass comprised of various compounds such as calcium, uric acid, struvite and/or cystine. Urinary tract stones include those comprised largely of calcium (calcium stone), uric acid (uric acid stone), stravite (stravite stone) or cystin e (cystine stone). It should be understood that each type of urinary tract stone can be comprised of numerous additional compounds in addition to calcium, uric acid, struvite, and cystine. In some embodiments, the urinar tract stone comprises zinc as the additional compound. Accordingly, in some embodiments, the urinary tract stone comprises calcium and zinc. According to the present invention, the reduction of the stone forming bioavailability of a zinc ion can be achieved via any method. In some embodiments, the stone forming bioavailability of zinc ions is reduced by administering to the subject a pharmaceutically effective amount of a composition comprising a zinc chelating agent, wherein the effective amount reduces the formation of a urinary tract stone in the subject. The term "zinc chelating agent" refers to a compound that binds a zinc ion and thereby reduces the stone forming bioavailability of the zinc ion. In preferred embodiments, the zinc chelating agent selectively binds a zinc ion. Non-limiting examples of zinc chelating agents are N,N,N',N'-Tetrakis-(2- pyridylmethyl)ethylenediamine (TPEN), disulfiram, cioquinol, phytic acid, and thioflavins.

In other or further embodiments, the stone forming bioavailability of zinc ions is reduced by administering to the subject a pharmaceutically effective amount of a composition comprising a composition that reduces zinc transport across a ceil membrane and thereby reduces the formation of a urinary tract stone in the subject. Any method known to those of skill in the art can be used to reduce zinc transport across a cell membrane. Accordingly, provided herein is a method of reducing the formation of a urinary tract stone in a subject in need of such reduction, comprising reducing transport of zinc ions by a cell in a tissue of the subject, thereby reducing the formation of the urinary tract stone. The cell can be, but is not limited to, a urinary tract cell, a kidney cell, or a kidney convoluted tubule cell.

An "upper urinary tract stone" can form in the kidney, whereas a "lower urinary tract stone" can form in the ureter or bladder as a result of crystal formation in the urine. Therefore, in some embodiments, the method of reducing the formation of an upper urinary tract stone in a subject in need of such reduction, comprises reducing transport of zinc ions in a kidney cell of the subject. In some of these embodiments, the upper urinary tract stone comprises calcium and zinc.

The renal tubules of the upper urinary tract comprise the proximal convoluted tubule, the

Loop of Henle, and the distal convoluted tubule. Accordingly, included herein is a method of reducing the formation of an upper urinary tract stone in a subject in need of such reduction, comprising reducing transport of zinc ions in a proximal convoluted tubule cell, a Loop of Henle cell, and/or a distal convoluted tubule cell of the subject. In some of these embodiments, the upper urinary tract stone comprises calcium and zinc.

As mentioned above, in some embodiments, a method of reducing the formation of a urinary tract stone in a subject in need of such reduction, comprises administering a pharmaceutically effective amount of a zinc chelator to the subject. In other or further embodiments, a method of reducing the formation of a urinary tract stone in a subject in need of such reduction, comprises reducing transport of a zinc ion across a ceil membrane. As used herein, the term "reducing transport of a zinc ion" includes both reducing the uptake and the export of a zinc ion from a cell, Tn some embodiments, the export of a zinc ion from a cell is reduced. In other embodiments, the import of a zinc ion into a ceil is reduced. In still other embodiments, the export and import of a zinc ion by a cell is reduced. It should be understood that "transport of a zinc ion by a cell" refers to the movement of zinc ions across a cell's membrane, or in other words, movement of zinc ions into or out of a cell. The cell can be, but is not limited to, a urinary tract cell, a kidney cell, or a kidney convoluted tubule cell.

In some embodiments, a method of reducing the formation of a urinary tract stone in a subject in need of such reduction, comprises reducing transport of a zinc ion across a cell membrane by reducing expression of a zinc transporter polynucleotide in a cell of the subject. As used herein, a "zinc transporter polynucleotide" refers to a polynucleotide (i.e., gene) that encodes a zinc transporter polypeptide. A "zinc transporter polypeptide" refers to a polypeptide involved in the transport of zinc ions across a cell membrane. Non-limiting examples of zinc transporter polypeptides include those polypeptides in the ZnT family and ZIP family of zinc transporters. ZnT proteins contribute to the cytoplasmic zinc balance by exporting zinc out to the extracellular space or by sequestering cytoplasmic zinc in intracellular compartments when cellular zinc levels are elevated. ZIP proteins function to increase cytoplasmic zinc concentrations when cellular zinc is depleted.

The ZnT family of genes currently comprises ten members: SLC30A1 (ortholog of D. melanogaster CG17723), SLC30A2 (ortholog of D. melanogaster CG3994), SLC30A3, SLC30A4 (ortholog of D. melanogaster CGI 1 163), SLC30A5, SLC30A6, SLC30A7, SLC30A8, SLC30A9, and SLC30A10. In some embodiments, a method of reducing the formation of a urinary tract stone in a subject in need of such reduction, comprises reducing expression of a SLC3GA1 , SLC30A2, SLC30A3, SLC30A4, SLC30A5, SLC30A6, SLC30A7, SLC30A8, SLC30A9, and/or SLC3GA10 gene in a cell of the subject by a therapeutically effective amount. In one embodiment, a method of reducing the formation of a urinary tract stone in a subject in need of such reduction, comprises reducing expression of a SLC30A2 gene in a kidney cell of the subject by a therapeutically effective amount. In still another embodiment, a method of reducing the formation of a urinary tract stone in a subject in need of such reduction, comprises reducing expression of a SLC30A2 gene in a convoluted tubule kidney cell of the subject by a therapeutically effective amount. The ZIP family of genes currently comprises ten members: SLC39A1 , 8LC39A2, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SLC39A7, SLC39A8, SLC39A10, and SLC39A14. In some embodiments, a method of reducing the formation of a urinary tract stone in a subject in need of such reduction, comprises reducing expression of a SLC39A 1 , SLC39A2, SLC39A3, SLC39A4, SLC39A5, SLC39A6, 8LC39A7, SLC39A8, SLC39A10, and/or SLC39A14 gene in a cell of the subject by a therapeutically effective amount. In one embodiment, a method of reducing the formation of a urinary tract stone in a subject in need of such reduction, comprises reducing expression of a SLC39A I, SLC39A2, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SLC39A7, SLC39A8, SLC39A10, and/or SLC39A14 gene in a kidney cell of the subject by a therapeutically effective amount. In other or further embodiments, a method of reducing the formation of a urinary tract stone in a subject in need of such reduction, comprises reducing expression of a SLC39AL SLC39A2, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SLC39A7, SLC39A8, SLC39A10, and/or SLC39A14 gene in a convoluted tubule kidney cell of the subject by a therapeutically effective amount.

According to the present method, reducing expression of a zinc transporter polynucleotide in a cell of a subject can be achieved via any method known to one of ordinary skill in the art. In some embodiments, expression is reduced by administering to the subject a pharmaceutically effective amount of an RNAi specific for a zinc transporter polynucleotide.

The term "RNA interference" sometimes called R A-mediated interference, post- transcriptional gene silencing, or quelling) refers to a phenomenon in which the presence of RNA, typically double-stranded RNA, in a cell results in inhibition of expression of a gene comprising a sequence identical, or nearly identical, to that of the double- stranded RNA. The double-stranded RN A responsible for inducing RN A interference is called an "interfering RN A" or "RNAi." Expression of the gene is inhibited by the mechanism of RNA interference as described below, in which (he presence of the interfering RNA results in degradation of mRNA transcribed from the gene and thus in decreased levels of the mRNA and any encoded protein.

The mechanism of RNA interference has been and is being extensively investigated in a number of eukaryotic organisms and cell types. See, for example, the following reviews: McManus and Sharp (2002) Nature Reviews Genetics 3:737-747; Hutvagner and Zarnore (2002) Curr Opin Genet & Dev 200:225-232; Hannon (2002) Nature 41 8:244-251 ; Agami (2002) Curr Opin Chem Biol 6:829-834; Tuschl and Borkhardt (2002) Molecular Interventions 2: 158-167; Nis kura (2001 ) Cell 107:415-41 8; and Zarnore (2001) Nature Structural Biology 8:746-750. RNA interference is also described in the patent literature; see, e.g., CA 2359180 by Kreutzer and Limmer; WO 01/68836 by Beach et al; WO 01/70949 by Graham et al; and WO 01/75164 by Tuschl et al.

In brief, double-stranded R A introduced into a cell (e.g., into the cytoplasm) is processed, for example by an RNAse Ill-like enzyme called Dicer, into shorter double-stranded fragments called small interfering RNAs (siRNAs, also called short interfering RNAs). The length and nature of the siRNAs produced is dependent on the species of the cell, although typically siRNAs are 21-25 nucleotides long (e.g., an siRNA may have a 19 base pair duplex portion with two nucleotide 3' overhangs at each end). Similar siRNAs can be produced in vitro (e.g., by chemical synthesis or in vitro transcription) and introduced into the cell to induce RNA. interference. The siRNA becomes associated with an RNA-induced silencing complex (RISC). Separation of the sense and antisense strands of the siRNA, and interaction of the siRNA antisense strand with its target mRNA through complementary base-pairing interactions, optionally occurs. Finally, the mRNA. is cleaved and degraded.

Expression of a target gene in a ceil can thus be specifically inhibited by introducing an appropriately chosen double-stranded RNA into the cell. Guidelines for design of suitable interfering RNAs are known to those of skill in the art. For example, interfering RNAs are typically designed against exon sequences, rather than introns or untranslated regions. Characteristics of high efficiency interfering RNAs may vary by cell type. For example, although siRNAs may require 3' overhangs and 5' phosphates for most efficient induction of RNAi in Drosophila cells, in mammalian cells blunt ended siRNAs and/or RNAs lacking 5' phosphates can induce RNAi as effectively as siRNAs with 3' overhangs and/or 5' phosphates. As another example, since double-stranded RNAs greater than 30-80 base pairs long activate the antiviral interferon response in mammalian cells and result in non-specific silencing, interfering RNAs for use in mammalian cells are typically less than 30 base pairs . The sense and antisense strands of a siRNA are typically, but not necessarily, completely complementary to each other over the double-stranded region of the siRNA. (excluding any overhangs). The antisense strand is typically completely complementary to the target mRNA over the same region, although some nucleotide substitutions can be tolerated (e.g., a one or two nucleotide mismatch between the antisense strand and the mRNA can still result in RNAi, although at reduced efficiency). The ends of the double-stranded region are typically more tolerant to substitution than the middle; for example, as little as 15 bp (base pairs) of complementarity between the antisense strand and the target mRNA in the context of a 21 mer with a 19 bp double-stranded region has been shown to result in a functional siRN A. Any overhangs can but need not be complementary to the target mRNA; for example, TT (two 2'-deoxythymidines) overhangs are frequently used to reduce synthesis costs.

Although double-stranded RNAs (e.g., double-stranded siRNAs) were initially thought to be required to initiate RNA interference, several recent reports indicate that the antisense strand of such siRNAs is sufficient to initiate RNAL Single-stranded antisense siRNAs can initiate RNA interference through the same pathway as double-stranded siRNAs (as evidenced, for example, by the appearance of specific mRNA endonucleolytic cleavage fragments). As for double-stranded interfering RNAs, characteristics of high- efficiency single -stranded siRNAs may vary by cell type (e.g., a 5' phosphate may be required on the antisense strand for efficient induction of RNA interference in some cell types, while a free 5' hydroxy! is sufficient in other cell types capable of phosphorylating the hydroxy!).

It should also be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims. All patents, patent applications, and publications referenced herein are incorporated by reference in their entirety for all purposes.

EXAMPLES

EXAMPLE 1

Creation of XDH knockdown flies

The Drosophila Malpighian tubule is the functional equivalent of the hitman kidney convoluted tubule. It is the site of solute transport and excretion of calcium, uric acid, and phosphorus, all of which are also regulated by the human renal tubule [Jarup, L. & Elinder, C. G. ( 1993) Br. J. Ind. Med. 50, 598-602], Ectopic calcification in the human kidney is an example of a pathologic human biomineralization process [Bagga, H. S. et al. (2013) Urol. Clin. North Am. 40, 1-12]. Given its homologous nature to human kidneys, we utilized the Drosophila Malpighian tubule to understand the initiating factors in biomineralization.

Based on protein sequence homology to genes known to affect stone formation in humans, candidate fly genes (Fig. 1 ) were selected to identify genetic mutations resulting in increased concretion formation in Malpighian tubules. Each transgenic fly strain containing inducible UAS-hairpin RNA. elements on their chromosomes towards a single coding protein sequence was purchased from the Vienna Drosophila RNAi Center, Vienna (VDRC), Austria. For total RNA and cDNA preparation, 5 to 10 flies were anesthetized with CO?, which were immediately homogenized using a Kontes® Microtube Pellet Pestle® Rods with Motor in the tube containing 350 μΐ RLT media supplemented with 3,5 μΐ of β-mercaptoethanol (RNeasy Mini Kit, Qiagen, Valencia, CA, USA) and total RNAs were isolated according to the manufacture's protocol. After measuring total RNA concentration and confirming their quality by examining their absorban.ce at 260 and 280 nm with DanoDrop (Thermo Scientific, Wilmington, DE, USA), cDNA were converted from the total RNAs using QuantiTec® Reverse Transcription Kit (Qiagen, Valencia, CA, USA). Using cDNA as a template, qPCR with SensiFast SYBR NO-Rox Kit (Taunton, MA, USA) was performed using a Light Cycler® 480 Real-Time PCR System (Roche Applied Science, Indianapolis, IN, USA) under following conditions: 95 °C, 5 seconds for denaturation, 55 °C, 20 seconds for annealing and extension. The specificity of amplicons was verified with a melting curve analy sis and the messenger levels were normalized using β-tubulin as an internal control and calculated according to the AACt method.

Virgin Daiighlerless(Da)-GAL4 flies were crossed into each transgenic male fly, then male progeny were sorted 14 days after initial crossings and transferred to low and high yeast diets. Tubules from adult male knockdown candidate flies were dissected and concretion formation determined on day 3 after sorting. In some instances, Da-GAL4 virgin flies were crossed into male VDRC lines containing a UAS-hairpin RNAi construct against a coding sequence of the xanthine dehydrogenase (XDH) gene (CG7642, VDRC transformant ID 25175). Ten days later, virgin progeny were collected, which were crossed again into male Tm6/Tm3 flies (courtesy of Haig Keshishian), a line that contains balancers on its third chromosome. Since all transgenes are located on the 3 ^U" chromosome, chromosomal recombination events during meiosis generated Da-GAL4, UAS-XDH RNAi /Tm6, which were easily identifiable according to eye color (dark red due to the knockdown of XDH) and the dominant marker Stubble from Tm6. The efficiency of chromosomal recombination was approximately 0.016%. The established recombinant line was utilized for all experimental assays.

Recombinant flies were anesthetized by C0 ₂ o standard flypads (Cat # 59-108, Genesee Scientific, San Diego, CA) and fly tubules were dissected under a dissecting light microscope (SZ61, Olympus, Center Valley, PA) on a culiure dish in droplets of Schneider's Drosophiia medium (Cat # 11720-034, mvitrogen, Carlsbad, CA), utilizing fine forceps (Roboz ceramic, Gaithersburg, MD). Tubules were imaged utilizing a Leica Ml 65 FC microscope with Leica Application Suite V3 analyzing software (Buffalo Grove, IL).

The level of concretion accumulation was quantified using the public domain linage.} software (http://rsb.nifo.nih.gov/ij). From images of dissected tubules captured with the Leica Ml 65 FC microscope, the tubules of interest were outlined, then total tubule area and pixel intensity histogram plots were obtained. Areas containing concretions were defined by setting the discrimination threshold to more than 3 standard deviations greater than the mean pixel intensity of wild-type flies (\v! !18). Any area in tubules whose pixel intensity was above the threshold was considered to be a concretion. Accordingly, the percentage of the tubule lumen area occupied by concretion was calculated. For all image analysis, background intensity was subtracted.

It was observed that RNAi silencing of xanthine dehydrogenase (XDH) [Fink, H. A. et al. (2009) European Urology 56, 72-80] resulted in increased tubule concretion formation when compared to wild type controls (data not shown).

EXAMPLE 2

High protein diet increases concretion formation in flies

As ectopic calcification accumulation in coronary artery disease, urinary stone formation, and osteoporosis is in part affected by diet [Heise, C. C. et al. (1988) Diabetes Care 1 1, 780-786; Evan, A. P. et al. (2008) Semiii. Nephrol. 28, 11 1-1 19; Evan, A. P. (2010) Pediatr Nephrol 25, 831-841], it was determined whether concretion formation in Drosophiia could be manipulated with dietary change. Upon XDH inhibition (Da-GAL4, UAS-XDH RNAi/+\ flies fed initially on standard laboratory food formed significantly more concretions after being transferred to a high protein diet (5% yeast extract (YE)) compared to a low protein diet (0.5% YE) (Figure 2.B). More specifically, male XDH knockdown (+/Da-GAL4; +/UAS-XDH RNAi) flies were reared on a normal diet (1.5% YE) till day 1 (left panel), and then transferred to either rich diet (5% yeast) (Figure 2A, middle panel) or dietary restriction condition (0.5% yeast) (Figure 2A, right panel). Two days later (day 3), the level of stone formation in each group was quantified. Under light microscopy examination, concretions were visible as dark intraluminal contents within the Malpighian tubule. The degree of mineralization was quantified by calculating the percentage of the lumen occupied by concretions.

Figure 2B demonstrates that following XDH knockdown, high protein feeding resulted in a 70% increase in Malpighian tubule concretion formation compared to low protein feeding. More specifically, flies with XDH knockdown (black bars) fed on high protein diet containing 5% YE (5% protein) were compared to those fed a low protein diet containing 0.5% YE (0.5% protein). The XDH knockdown flies developed statistically significantly greater stone formation (p<0.001 ). These results identified a gene knockdown candidate with a strong, modifiable phenotype which was utilized for further study.

EXAMPLE 3

Malpighian tubule concretions comprise calcium, zinc and magnesium

To analyze the chemical nature of the Malpighian tubule concretions, a fluorescently- labeled bisphosphonate dye was injected into the abdominal cavity of flies upon XDH knockdown. More specifically, to obtain sufficient stones for ICP-OES, .i XRF, μΧΚΙΙ, and μΧΑΝΕ-S analysis, Malpighian tubules were dissected in sterile water and transferred to a pre- weighed centrifuge tube and sonicated (Ultrasonic Processor GEX130 sonicator, Cole-Parmer. Instruments, Vernon Hills, IL) at an amplitude of 20 for 5 seconds to separate tubule tissue from intraluminal concretions. After concretions settled by gravity for 10 minutes, the supernatant was removed and the concretions were washed 3 times in sterile water to remove remaining tubule tissue. Water was removed by aspiration and the concretions were allowed to dry in a negative pressure hood, which gave rise to approximately 1 mg of concretions from 150 Da- GALA, UAS-XDH RNAi/+ flies fed on a high protein diet for 2 days. A bisphosphonate dye bound to a tluorophor was synthesized to examine the presence of hvdroxyapatite in fly stones [Yepiskoposyan, H. et al. (2006) Nucleic Acids Res. 34, 4866-4877]. The dye was manually micro-injected into abdominal cavity of flies and 18 hours after injection, tubules were dissected and examined using indirect fluorescent microscopy utilizing a Leica Ml 65 FC microscope to localize the dye distribution. After 24 hours, avid fluorescent staining of the intraluminal concretions within the tubule was observed (Fig. 3A).

Bisphosphonates are known to bind avidly to calcium salts [Russell, R. G. G. (201 1) Bone 49, 2-19], and therefore micro X-ray absorption near edge spectroscopy (liXANES) was used to confirm that hydroxyapaiite was present as a binding target (Fig. 3B). μΧΚΡ mapping and μΧΑ-NES measurements were performed on beamiine 10.3.2 of the Advanced Light Source at Lawrence Berkeley National Lab (Berkeley, CA). Fly concretions and Randall's plaques were mounted onto X-ray transparent 100 nm thick Si3N4 windows (Silson Ltd). Maps were collected at I l k eV using a 3 x 3 μνα beam spot size, 2 x 2 μηι ixels, 150 ms dwell / ^' pixel. Ca K-edge and Zn K-edge spectra were collected in QXAS mode on Randall's plaque and Da- GA.L4, UAS-XDH RNAU+ concretions. Spectra were dead-time corrected, pre-edge background substracted and post-edge normalized using standard procedures [Blaschko, S. D. et ai. (2013) J. Urol. 189, 726-734]. Spectra were calibrated using an Sb foil (4132.2 eV), Least-square linear combination fitting the spectra was performed using a database of standards. Concretions were also analyzed with powder micro-x-ray diffraction ^uXRD), demonstrating the presence of crystalline structures within each sample (Fig. 3C).

Further, inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to demonstrate calcium (Ca ² ), magnesium (Mg ^{2 '} ), and zinc (Zrr ) as the major metal elements present in each specimen (Fig. 3D). More specifically, stone elemental analysis was determined by inductively coupled plasma - optical emission spectroscopy (ICP-OES). Isolated and pelleted dry concretion samples in afiquots of 500 }tg were dissolved in OmniTrace 70% H 03 (VWR Scientific) and then diluted with OmniTrace water to 5% HN03 before being transferred via pneumatic nebulizer into a Vista Pro ICP (Varian). Elemental values were calibrated by using National Institute of Standards and Technology (NIST)-traceahie standards. Cesium was used for ionization suppression and yttrium was used as an interaal standard. Data were collected and analyzed using native software (ICP Expert). Three of 33 detectable elements were measured using conventional minimal detection limits for each element. The presence of hydroxyapaiite, crystalline particles, and minerals suggest that Drosophila concretion formation is representative of many biomineralization processes [Pearle, M. et al. (2005) J. Urol. 173, 848-857; Randall, A. (1937) Ann. Surg. 105, 1009-1027: Ryail, R, L. (2008) Urol. Res. 36, 77-97]. EXAMPLE 4

Randall's plaques comprise non-trace amounts of zinc

With bisphosphonate dye, it was shown that Drosophila fly stones contain apatite crystals (Figure 3 A), a common component of human urinary stones, and therefore may undergo a similar process of biominera!ization. To compare Drosophila fly stone material to a process of biomineralization in human tissue, human renal papilla biopsies containing Randall's plaque material were used for comparative analyses. Randall's plaques are calcified structures residing in the tip of the renal papilla that are thought to be precursor lesions to kidney stones [Randall, A. (1937) Ami. Surg. 105, 1009-1027] and one example of ectopic mineralization resulting in disease [Bagga, H. S. et al. (2013) Urol. Clin. North Am. 40, 1 -12]. For human Randall's plaque studies, after appropriate institutional human research protocols were approved, renal papilla biopsies w ere taken from kidneys during surgery for removal of urinary stones.

Gross photomicrographs were taken of Malpighian tubule concretions from flies after XDH inhibition and Randall's plaques isolated from human renal papillary biopsies (Fig. 4A). Transmission electron microscopy imaging further revealed the presence of similar ring-like structures in both samples (Fig. 4B, upper and lower panel). These ring structures also have been observed in electron microscopic imaging of forming bone, atherosclerotic plaques, and other forms of ectopic calcification [Bobryshev, Y. V. et al. (2008) J. Cell Mol. Med. 12, 2073-2082; Kajander, E. O. & Ciftcioglu, N. (1998) Proc. Natl. Acad. Sci. USA 95, 8274-8279] and may represent a fundamental building block in biomineralization [Martel, J. & Young, J. D.-E. (2008) Proc. Natl. Acad. Sci. USA 105, 5549-5554], However, given their small size of approximately 100 ran in diameter, they have been poorly characterized in the prior art

For transmission electron microscopy, dissected Malpighian tubules containing concretions were immersed in buffered aldehyde, stained with osmium terroxide and then en bloc with uranyl acetate, and embedded in Embed 812 (Electron Microscopy Sciences, Hatfield, PA). 100 urn sections were collected on slot grids, post-stained with uranyl acetate and lead citrate, and viewed on a Philips Tecnai 12 electron microscope. Human Randall's plaque samples were fixed in 2% paraformaldehyde and 2.5% giutaraidehycie in 0.150 M sodium cacodylate buffer for 14 hours, then rinsed in 0.1 M sodium cacodylate buffer and post-fixed in 2% osmium tetroxide and 0.8% potassium ferrocyanide in 0.1 M sodium cacodylate for 60 minutes. After rinsing and staining with 2% uranyl acetate for 30 minutes, dehydration was performed using a series of ethanol dilutions followed by submersion in 100% ethanoi and infiltration with propylene oxide followed by Epon-812. Samples were embedded in epon-filled beem capsules and allowed to polymerize at 60°C for 72 hours. Blocks were sectioned using a MT-7000 ultramicrotome (KMC Products, Boeckeler Instruments, Tucson, AR) at 60 nm and imaged on a Phillips Tecnai 12 transmission electron microscope.

Finally, Drosophiia concretions and hitman Randall's plaques were analyzed using micro X-ray fluorescence ^XRF) mapping to determine if their compositions were similar. μΧΡί mapping at l lkeV with 15 micron / pixel and 2.5 micron / pixel resolution demonstrated the consistent presence of both Ca ²⁺ and Zn ^z" in each specimen (Fig. 4C). μΧΚΡ spectra recorded at 14 keV of randomly selected regions of the samples demonstrated an abundance of Ca ^{2 :} and Zn ^2" , with trace amounts of iron also present (Fig. 4D). The presence of non-trace amounts of Zn ^2, in both tissue samples was surprising and was targeted for further investigation.

EXAMPLE 5

Zinc is a critical initiator for stone formation in Drosophiia

The role of Zn ^: in initiating biomineraliza ion in Drosophiia was determined using dietary, pharmacologic, and genetic manipulations. More specifically, adult male flies were selected from a laboratory colony and maintained on standard fly yeast extract medium (1.5 % YE) containing 1.5 g of yeast, 5 g of sucrose, 0.46 g of agar and 8.5 g of com meal in 100 ml distilled water. For high protein (5 % YE) and low protein (0.5 % YE) diets, the amount of yeast was adjusted to 5 g and 0.5 g, respectively. Propionic acid was used to prevent moid growth. For survivorship analysis, fly survival was scored while flies were transferred to fresh media every 2-3 days and the significance of change was measured using the fog-rank test as previously- described [Blaschko, S. D. et al. (2013) J. Urol. 189, 735-739]. All flies were obtained or purchased from Bloomington Drosophiia Stock Center (Indiana, IN) and VDRC (Vienna, Austria) unless otherwise mentioned. Flies were kept in a temperature (25°C) and humidity (60%) controlled designated fly room with a 12-hour light / dark cycle.

After two days, adult male flies were dissected and their tubules imaged for the presence of concretions. Supplementing a low protein diet with 1 mM and .10 mM Z11SO4 yielded increased concretion formation in a dose-dependent fashion compared to a low protein diet without Ζΐί" supplementation (Fig. 5A). ICP-OES analysis of whole flies demonstrated an increase in Zn^ levels less than that expected based on the dietary Zn ^{2 i} content (data not shown). Based on this observation, it was reasoned that Zn ^: sequestration and transport was a critical factor in facilitating concretion formation. To verify this relationship more directly, the transport of Zrr ^: was blocked using a pharmacologic approach to demonstrate that reduction of bioavailable Zn ^l would have an opposite effect on concretion formation compared to Zn ²⁺ feeding. Supplementation with N,N, ' '-Tetrakis-(2-pyridyfmethy])ethylenediamine (TPEN), a zinc chelator [Li, C, et al. (2010) J. Bone Miner. Res. 25, 968-975; Landi, E. et al. (2007) Acta Biomater. 3, 961-969], resulted in reduction of concretion formation in flies fed a high protein diet after XDH inhibition (Fig. 5B). A 100 μΜ concentration of TPEN was selected as the non- lethal dose that demonstrated the maximal effect on suppressing concretion formation (data not shown). The protective effect of TPEN could be overcome with Zn ^" , but not with Mg" supplementation (Fig. 5B), demonstrating the specificity of TPEN for Zn ⁺ on concretion formation.

Genetic manipulation by suppressing expression of members of the ZnT family of genes tested the effects of Zn ^{2 r} transport. Zn ^? " transport was inhibited using RNAi against three individual Zn ⁺ transporters, CGI 1163, CGI 7723 and CG3994 in Drosophila with suppressed levels of XDH. Of these three Zn ^{2 ;} transporters, CGI 7723, and CG3994 have been functionally characterized and reported to be highly expressed in the gut and Malpighian tubule [Ferraro, P. M. et ai. (201 1) J. Endourol. 25, 875-880; Roh, H. C. et al. (2012) Cell Metabolism 15, 88-99], RNAi knockdown efficiency was confirmed with qPCR (data not shown). Simultaneous inhibition of XDH and each of the Zn ² transporter genes reduced concretion formation (Fig. 5C). These results demonstrated reduction in mineralization by altering Zn ^{2 "} transport. When fed a high protein diet, concurrent knockdown of XDH with each Zn ⁺ transporter increased survivorship compared with XDH inhibition alone, where survivorship was dramatically shortened (Fig. 5D).

As CG3994 is exclusively expressed in the apical portion of the Malpighian tubule [Ferraro, P. M. et al. (2011) J. Endourol. 25, 875-880] and was the Zn ^z+ transporter demonstrating both the strongest reduction in concretion formation and rescued survivorship (Fig. 5C and 5D), it was selected for further investigation. Upon simultaneous XDH and CG3994 inhibition in Drosophila, lCP-QES analysis revealed that whole-fly Zn ^{2 i} levels were increased in a specific fashion while other cation levels remained unchanged, suggesting this transporter may occupy an important role in facilitating Zn ²⁴ tubule excretion (data not shown).

Together, dietary Z ^Zn supplementation, Zn ^; -specific chelation with TPEN, and ZnT transporter knockdown demonstrated a central role for Zn ^{2 h} in concretion formation. In microealcifications of early atherosclerotic plaques, zinc has been co-localized to areas of high calcium concentration that contain hydroxyapatite [Wang, X. et al. (2009) FASEB J. 23, 2650- 2661]. However, the role of zinc in initiating the formation of mineralized tissue has remained largely uncertain. It is demonstrated herein that hydroxyapatiie and Zn ² appear together in both Drosophila Maipighian tubule concretions and human renal papillary Randall's plaques, two mineralized tissues across two different species. This indicates that Zn" ¹ likely plays a critical role in initiating biomineralization since hydroxyapatiie is thought of as an early nidus for calcification. Thus, manipulation of Zn ^2' can be leveraged as a therapeutic target for modulation of pathologic biomineralization processes.

EXAMPLE 6

Drosophila as a translational model for human urinary tract stone formation

Provided herein is data establishing D. melanogasier as a translational model to study nephrolithiasis. Significant similarities in the excretory system and stone formation between flies and humans demonstrated herein provides insights into the biological basis of human stone formation. Briefly, the similarities between the fly and humans include, but are not limited to, the following: 1) Functional similarity in the ion transport properties of the Maipighian tubule to the human kidney [Dow, J. T, & Davies, S. A. (2003) Physiol. Rev. 83, 687-729]; 2.) The anatomic connection of the Maipighian tubules with the ureter that displays peristaltic activity is reminiscent of the human ureter; 3) Observed conservation of genes in the Maipighian tubule and in humans [Dow, J. A. T. & Davies, S. A. (2006) J. Insect. Physiol 52, 365-378]; 4) Conservation of over two hundred human disease genes including many involved in kidney dysfunction in the fly; 5) Identification of the fly ortholog of xanthine dehydrogenase, which causes stone formation in flies and humans; 6) Biochemical similarities between stones in the fly with those in humans including the presence of calcium, xanthine, and an affinity for an apatite- binding bisphosphonate dye; 7) Structural similarity between human and fly stones using electron microscopy; 8) The importance of a high protein diet in stone fonnation in both flies and humans; and 9) Identification of a critical and conserved role for zinc in stone formation.

Accordingly, it is demonstrated herein that Drosophila is an excellent translational model for urinary stone disease. The ability to singly inhibit transporters permitted development of a genetically based, controlled model for stone disease in the fly which exhibited production of tubule stones of similar structure and composition compared to human urinary stones. Analysis of these stones demonstrated the presence of zinc. After confirming the presence of zinc in human urinary stones, this finding back was applied to a fly model to test whether these metals could be leveraged as therapeutic targets. Together, dietary Zn supplementation, Zn-specific chelation with TPEN, and ZnT transporter knockdown demonstrated a central role for Zn in stone formation. In microcalcifications of early atherosclerotic plaques, zinc has been co-localized to areas of high calcium concentration that contain hydroxy apatite [Roijers, R. B, et al. (2011) Am. J. Pathol. 178, 2.879-2887], Zinc is also know to facilitate osteoblastic activity while inhibiting osteoclastic activity in bone [Kawakubo, A. et al. (201 1) Microsc. Res. Tech. doi: 10.1002/jemt.21009] . However, the role of zinc in initiating the formation of mineralized tissue has remained largely uncertain.

Provided herein are findings that hydroxyapatite and Zn appear together in both

Drosophi!a Malpighian tubule stories and human renal stones, two mineralized tissues across two different species, and that the modulation of Zn led to changes in stone formation in the fly model. Thus, manipulation of Zn can be leveraged as a therapeutic target for the treatment of nephrolithiasis.