WO2005097176A2 - Modulating oxalate transport - Google Patents

Abstract

Compositions and methods for increasing the rate of oxalate transport from a parenteral site to the intestinal lumen in an animal subject are described herein. The compositions and methods are useful for treating an animal subject suffering from renal failure. By increasing the number of Oxalobacter bacteria or Oxalobacter enzymes in the animal subject, oxalate is transported to and degraded in the intestinal lumen, thereby reducing the burden of oxalate excretion by the kidneys.

Description

MODULATING OXALATE TRANSPORT

FIELD OF THE INVENTION The invention relates to compositions and methods for treatixig animal subjects, including humans, suffering from renal disease with Oxalobacter sp. bacteria or Oxalobacter enzymes.

BACKGROUND Oxalic acid is a toxic by-product of metabolism and a comm-on constituent of most diets. Over production of oxalic acid can result in a number of pathological conditions, e.g., hyperoxaluria, hyperoxalemia, calcium oxalate nephrolithiasis, and oxalosis leading to cardiomyopathy and cardiac conductance disorders. In addition, several pathological conditions, including Crohn's disease, steatorrhea, kidney disease resulting from jejuno-ileal bypass surgery and cystic fibrosis, are associated with enteric hyperoxaluria due to enhanced oxalic acid absorption in the gastrointestinal tract. Evidence indicates that Oxalobacter formigenes maintains an important symbiotic relationship with its hosts by regulating oxalic acid homeostasis. Several studies have shown that O. formigenes may influence several oxalate-related diseases, since the absence of the bacterium correlates with hyperoxaluria and episodes of kidney stone formation. More notably, however, colonization with O. formigenes significantly lowers hyperoxaluria in rats fed a diet rich in oxalate. Thus, lack of colonization of the GI tract with O. formigenes appears to be an additional risk factor for calcium oxalate urolithiasis. Since the most promising treatment available for primary hyperoxaluria is a combined kidney-liver transplant, and the treatment for idiopathic oxalate stone disease is based upon generalized dietary restrictions and increased fluid intake, there is otrviously an insistent need in the art for effective alternative management options. SUMMARY The invention relates to the discovery that the actions of O. formigenes and/or lysate- enzyme preparations thereof promote an enhanced enteric elimination of oxalate. This enteric excretion was notably higher for animals suffering from chronic renal failure than for animals with normal renal function. The administration of O. formigenes, or products thereof, can be used to enhance the extra-renal elimination of oxalate from the circulatory system via the intestines, thereby reducing the burden of oxalate in the kidney. Lowering systemic oxalate levels through the use of O. formigenes could prove to be of significant benefit to patients suffering from renal diseases, such as chronic renal failure (CRF). In a preferred embodiment, the method of increasing the rate of oxalate transport from a parenteral site to an intestinal lumen in an animal subject suffering from renal failure comprises administering to the subject a composition comprising oxalate substrate specific Oxalobacter; thereby, increasing the number of oxalate-degrading bacteria or amount of oxalate-degrading bacteria enzymes in the gastrointestinal tract in the animal. Preferably, the Oxalobacter bacteria colonize the intestines, locally stimulate active transport systems involved in colonic oxalate secretion and, also stimulate intraluminal oxalate-degradation. Preferably, the oxalate degradation produces an outwardly directed concentration gradient and the active component has an oxalate degrading capacity between about 0.1 μmole to about 100.0 μmole oxalate/min/mg protein. In a preferred embodiment, the method of increasing the number of oxalate-degrading bacteria or amount of oxalate-degrading bacteria enzymes in the gastrointestinal tract in the animal comprises administering a composition comprising Oxalobacter to the animal subject. Preferably, the oxalate-degrading bacteria is Oxalobacter formigenes and the bacteria can be viable and/or a lysate thereof. In a preferred embodiment, the composition comprises a pharmaceutically acceptable carrier that is resistant to degradation by gastric acidity. The composition can be in tablet form and/or paste form. In another preferred embodiment, the subject is a mammal wherein the renal failure is associated with oxalate levels in the kidneys. In accordance with the invention the patient is suffering from hyperoxaluria. In another preferred embodiment, the invention provides a method of reducing the amount of time that a subject suffering from renal failure must spend on dialysis, the method comprising the step of administering to the subject a composition comprising Oxalobacter bacterium or Oxalobacter enzymes, wherein administration of the composition results in decreased levels of oxalate excretion. In another preferred embodiment, a pharmaceutical composition comprises a lysate/enzyme preparation of Oxalobacter bacterium and Oxalobacter enzymes, whereby, the composition is formulated to prevent degradation in acidic conditions. The acidic conditions are the conditions found in the stomach, and the composition can be encapsulated in a capsule which does not dissolve at 37° C at pH < 6.0. Preferably, the composition comprises a lysate of substrate-specific Oxalobacter bacteria and oxalate-degrading enzymes. The oxalate- degrading enzymes are preferably formyl-CoA transferase and oxalyl CoA decarboxylase. Preferably, the composition is formulated to provide an oxalate degrading capacity of between about 50 mg to about 900mg oxalate/day. Each dose of the composition comprises about 1.Omg to about 20 mg of active enzyme component with an oxalate degrading capacity between about 0.1 μmole to about 100.0 μmole oxalate/min/mg protein. In another preferred embodiment, a method of treating a patient suffering from renal disease comprises administering to the patient a composition comprising oxalate substrate specific Oxalobacter lysates and Oxalobacter enzymes, wherein administration of the composition increases the rate of oxalate transport from a parenteral site to the intestinal lumen. Preferably, the Oxalobacter lysates and Oxalobacter enzymes are in a ratio of 1 : 1 (v/v) up to a ratio of 1:50 (v/v). Preferably, the Oxalobacter lysate and Oxalobacter enzyme compositions are formulated to provide an oxalate degrading capacity of between about 50 mg to about 900mg oxalate/day. In accordance with the invention, these ratios can be manipulated according to the patient's response to the treatment. Therefore, in some patients the Oxalobacter lysates and Oxalobacter enzymes are in a ratio of 5:1 (v/v) up to a ratio of 50:1 (v/v). In a preferred embodiment, the method further provides administration of oxalate- degrading enzymes, such as, formyl-CoA transferase and oxalyl CoA decarboxylase. The enzymes can be administered before, during and/or after administration of the composition comprising Oxalobacter lysates and Oxalobacter enzymes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The particular embodiments discussed below are illustrative only and not intended to be limiting. Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is pointed out with particularity in the appended claims. The above and further advantages of this invention may be better understood by referring to the following description taken in conjunction with the accompanying drawings, in which: FIG. 1 is a graph showing a reduction of hyperoxaluria via increased secretion of oxalic acid across the intestinal wall. Grp I: non-treated, Grp II: ethylene glycol (EG) diet, Grp III: EG diet + O. formigenes, Grp IN: EG + oxalate diet + O. formigenes. FIG. 2 is a graph showing colonic oxalate transport in hyperoxaluric-induced CRF rats treated with encapsulated Oxalobacter lysate or placebo. FIG. 3 is a graph showing regulation of hyperoxaluria in the rat model via reduction of oxalic acid absorption across the intestinal wall. FIG. 4 is a graph showing colonic oxalate transport in naturally colonized rats with normal renal function. FIG. 5 is a graph showing that there were no significant effects of the membrane fragment preparation on oxalate transport across the distal colon (n=9 tissue pairs from 4 rats). FIG. 6 is a graph showing that addition of an aliquot of the heat-treated lysate (final protein concentration 0.54 mg/ml of mucosal bathing solution) induced a significant net secretion of oxalate across the distal colon (n=7 tissue pairs from 4 rats). FIG. 7 is a graph showing that similar to normal healthy control rats with both kidneys intact, the distal segment of the UN rats supports a significant basal net absorptive flux of oxalate. FIG. 8 is a graph showing that colonic oxalate transport was significantly altered in the rats receiving the Oxalobacter lysate compared to the placebo-treated rats.

DETAILED DESCRIPTION It has been discovered that there exists a relationship between intestinal secretion of oxalate and the presence of O. formigenes in the intestinal tract and that this axis plays a role in maintaining oxalate homeostasis. Local modulation of luminal oxalate activity by O. formigenes degradative action facilitates an outwardly directed oxalate gradient across the epithelial barrier. Oxalobacter is thought to derive its substrate, oxalate, by way of transmural passive and active pathways in oxalate-induced renal failure and the activity of this microorganism can potentially regulate and reduce the resulting luminal oxalate activity. A dynamic interaction exists between the capacity of the intestinal mucosal barrier to secrete and excrete oxalate coupled with the ability of Oxalobacter sp. to lower intraluminal oxalate activity. The invention provides methods and compositions for increasing the rate of oxalate transport from a parenteral site to the intestinal lumen in an animal subject suffering from renal failure. Studies described herein demonstrate that oxalate excretion into the lumen of the large, intestine, where it can be degraded innocuously by the local microflora, decreases oxalate levels in the urine. This enteric excretion of oxalate that bypasses the kidneys should have a significant impact on reducing hyperoxalemia, hyperoxaluria, oxalosis and the resulting various pathophysiological and debilitating conditions. The methods and compositions of the invention are particularly advantageous for preventing hyperoxaluria and oxalate crystal deposition in the tissue and the formation of kidney stones containing calcium oxalate.

Definitions "Diagnostic" or "diagnosed" means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who are not diseased and who test negative in the assay, are termed "true negatives." The

"specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. The terms "patient" or "individual" are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates. "Treatment" is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. As used herein, "ameliorated" or "treatment" refers to a symptom which approaches a normalized value (for example a value obtained in a healthy patient or individual), e.g., is less than 50% different from a normalized value, preferably is less than about 25% different from a normalized value, more preferably, is less than 10% different from a normalized value, and still more preferably, is not significantly different from a normalized value as determined using routine statistical tests. As used herein, a "pharmaceutically acceptable" component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. As used herein, the term "safe and effective amount" refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention. By "therapeutically effective amount" is meant an amount of a compound of the present invention effective to yield the desired therapeutic response. By the term "modulate," it is meant that any of the mentioned activities, are, e.g., increased, enhanced, agonized (acts as an agonist), promoted, decreased, reduced, suppressed blocked, or antagonized (acts as an agonist). Modulation can increase activity more than 1- fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., over baseline values. Modulation can also decrease its activity below baseline values. The below described preferred embodiments illustrate adaptations of these methods and compositions. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

Increasing the Rate of Oxalate Transport from a Parenteral Site to the Intestinal Lumen in an Animal Subject The invention provides a method for increasing the rate of oxalate transport from a parenteral site to the intestinal lumen in an animal subject suffering from renal failure. The method includes the steps of providing an animal subject with renal failure and increasing the number of Oxalobacter bacteria or amount of Oxalobacter enzymes in the gastrointestinal tract of the animal. An animal subject suffering from renal failure can be a subject having low levels of Oxalobacter within the intestines or a subject entirely lacking intestinally- residing Oxalobacter. Any suitable method for increasing the number of Oxalobacter bacterium or amount of Oxalobacter enzymes in the gastrointestinal tract in the animal may be used. For example, the Oxalobacter bacteria or Oxalobacter enzymes can be delivered to the gastrointestinal tract in an animal subject by orally administering a composition including Oxalobacter bacteria, Oxalobacter lysate, Oxalobacter enzymes, or nucleic acid encoding one or more Oxalobacter enzymes. The animal subject suffering from renal failure to which a composition including Oxalobacter bacterium or Oxalobacter enzymes is administered is preferably a mammal (e.g., a human). A preferred Oxalobacter species for increasing the rate of oxalate transport from a parenteral site to the intestinal lumen in an animal subject suffering from renal failure is O. formigenes, however, any oxalate-degrading bacteria can be used in compositions and methods of the invention. Examples of other oxalate-degrading bacteria include Clostridium, Bifidobacterium, Bacillus, and Pseudomonas. The oxalate-degrading bacteria (e.g.,

Oxalobacter) that are administered can be in the form of live organisms, as these are non- pathogenic bacteria normally inhabiting the guts of mammals. Portions of oxalate-degrading bacteria can also be administered, such as an Oxalobacter cellular lysate or isolated oxalate- degrading bacterial enzymes. Alternatively, a nucleic acid encoding one or more Oxalobacter enzymes can be administered to the subject. Nucleic acid (i.e., DNA) sequences encoding these enzymes are known to those skilled in the art and are described in, for example, WO 98/16632. In a specific embodiment, the subject invention pertains to the preparation and administration of cells of oxalate-degrading bacteria of the species, Oxalobacter formigenes, to the human or animal intestinal tract where the activity of the microbes reduces the amount of oxalate present in the intestine thereby causing a reduction of concentrations of oxalate in the kidneys and in other cellular fluids. The introduced cells degrade oxalate and replicate in the intestinal habitat so that progeny of the initial cells colonize the intestine and continue to remove oxalate. This activity reduces the risk for formation of kidney stones as well as other disease complications caused by oxalic acid. In a preferred embodiment for human use, the specific strains of O. formigenes used are isolates from human intestinal samples. The strains are thus part of the normal human intestinal bacterial flora. However, since they are not present in all persons, or are present in insufficient numbers, the introduction of these organisms corrects a deficiency that exists in some humans. Enrichment of the contents of the intestines with one or more species of oxalate- degrading bacteria causes a reduction of oxalate in the intestinal contents. Some of the bacteria carry out oxalate degradation at or near the site of absorption (herein referred to as "locally"). The activity of the bacteria decreases the level of absorption of dietary oxalate. A reduction in oxalate concentration in the intestines can also lead to a removal of oxalate from cells and the general circulation. More specifically, a reduction of oxalate concentration in the intestines can also lead to enhanced secretion of oxalate into the intestine from the blood and thus reduce the amount of oxalate that needs to be excreted in urine. Thus, the methods of the subject invention can be used to treat primary hyperoxaluria in addition to treatment of dietary hyperoxaluria. T he materials and methods of the subject invention are particularly advantageous in the promotion of healthy oxalate levels in humans and animals, thereby representing a possible dietary supplement for healthy individuals as well. In another preferred embodiment, the invention provides a pharmaceutical composition comprising a lysate/enzyme preparation of Oxalobacter bacterium and Oxalobacter enzymes, whereby, the composition is formulated to prevent degradation in acidic conditions. Preferably, the composition comprises a lysate of substrate-specific Oxalobacter bacteria and oxalate-degrading enzymes. The oxalate-degrading enzymes are preferably formyl-CoA transferase and oxalyl CoA decarboxylase. Without wishing to be bound by theory, the lysate/enzyme composition locally stimulates the active transport systems involved in colonic oxalate secretion. The lysate/enzyme composition's intraluminal oxalate-degradative capacity serves to sustain an outwardly directed concentration gradient such that transmural passive movement of oxalate from the blood into the lumen is also enhanced. Together, these independent actions of the Oxalobacter lysate/enzyme preparation can optimally promote enteric elimination of oxalate. Pharmaceutical compositions for the introduction of oxalate degrading bacteria and/or enzymes, lysate/enzyme preparations into the intestine include bacteria, bacterial lysates and/or enzymes that have been lyophilized or frozen in liquid or paste form and encapsulated in a gel capsule or other enteric protection. The gel cap material is preferably a polymeric material which forms a delivery pill or capsule that is resistant to degradation by the gastric acidity and enzymes of the stomach but is degraded with concomitant release of oxalate- degrading materials by the higher pH and bile acid contents in the intestine. T he released material then converts oxalate present in the intestine to harmless products. Pharmaceutical carriers also can be combined with the bacteria or enzymes. These would include, for example, saline-phosphate buffer. In another preferred embodiment, the invention also provides a method of reducing the amount of time that a subject suffering from renal failure must spend on dialysis. The method includes the steps of providing a subject suffering from renal failure and administering to the subject a composition including Oxalobacter bacteria or Oxalobacter enzymes. Administration of the composition results in decreased levels of oxalate excreted by dysfunctional kidneys in the subject. This method is particularly useful for patients on peritoneal dialysis. Subjects Because subjects from many different species are susceptible to hyperoxaluria and pathological conditions associated therewith (e.g., CRF), the invention is believed to be useable with many types of animal subjects. A non-exhaustive exemplary list of such animals includes mammals such as mice, rats, rabbits, goats, sheep, pigs, horses, cattle, dogs, cats, and primates such as monkeys, apes, and human beings, as well as laboratory and zoological animals. Those animal subjects known to suffer from hyperoxaluria and development of subsequent CRF are preferred for use in the invention. In particular, human patients suffering from primary hyperoxaluria and CRF are suitable animal subjects for use in the invention. In the experiments described herein, the subjects used were rats. Nonetheless, by adapting the methods taught herein to other methods known in medicine or veterinary science (e.g., adjusting doses of administered substances according to the weight of the subject animal), the compositions utilized in the invention can be readily optimized for use in other subjects.

Pharmaceutical Compositions and Administration to a Subject The compositions described above may be administered to animals including human beings in any suitable formulation. For example, an Oxalobacter composition (e.g., Oxalobacter cells, Oxalobacter enzyme(s), nucleic acid encoding one or more Oxalobacter enzymes) may be formulated in pharmaceutically acceptable carriers or diluents such as physiological saline or a buffered salt solution. Suitable carriers and diluents can be selected on the basis of mode and route of administration and standard pharmaceutical practice. A description of exemplary pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in Remington's Pharmaceutical Sciences, a standard text in this field, and in USP/NF. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the compositions maybe in powder form (e.g., lyophilized) for administration or for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. Preferably, the Oxalobacter cells, lysate, or enzymes, are in an encapsulated form suitable for oral administration. The capsule can be a gel capsule, for example. The compositions can also be administered in tablet, liquid, or paste form. Nucleic acids encoding Oxalobacter enzymes are expressed in a viral vector. The compositions of the invention may be administered to animals by any conventional technique. Typically, such administration will be oral. However, parenteral administration of the bacteria through suppositories (e.g., intra-anal introduction) or the cell- free enzymes (e.g., intraperitoneal introduction) is also -within the invention. The compositions may also be admimstered directly to a target tissue (e.g., the kidneys) by, for example, direct injection into, or surgical delivery to, an internal or external target tissue. The compositions can be delivered as capsules or microcapsules designed to protect the material from adverse effects of acid stomach. One or more of several enteric protective coating methods can be used. Descriptions of such enteric coatings include the use of cellulose acetate phthalate (CAP) (Yacobi, A., E. H. Walega, 1988, Oral sustained release formulations: Dosing and evaluation, Pergammon Press). Other descriptions of encapsulation technology include U.S. Pat. No. 5,286,495, which is incorporated herein by reference. Strains of Oxalobacter useful according to the subject invention have been characterized based upon several tests as described in detail in the Examples which follow. Other methods include: patterns of cellular fatty acids, patterns of cellular proteins, DNA and RNA (Jensen, N. S., M. J. Allison (1995) Abstr. to the General Meeting of the Amer. Soc. Microbiol., 1-29), and responses to oligonucleotide probes (Sidhu et al. 1996). One embodiment of the present invention involves procedures for selection, preparation and administration of the appropriate oxalate-degrading bacteria, bacterial lysate/enzyme preparations to a diversity of subjects. Prominently, but not exclusively, these are persons or animals which do not harbor these bacteria in their intestines. These non- colonized or weakly-colonized persons or animals are identified using tests that allow for rapid and definitive detection of Oxalobacter even when the organisms are at relatively low concentrations in mixed bacterial populations such as are found in intestinal contents. The methods of the subject invention can also be used to treat individuals or animals whose oxalate-degrading bacteria have been depleted due to, for example, antibiotic treatment or in post-operative situations. The methods of the subject invention can also be used to treat individuals or animals who have colonies of oxalate-degrading bacteria but who still have unhealthy levels of oxalate due to, for example, oxalate susceptibility and/or excessive production of endogenous oxalate. Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Agents intended to be administered intracellularly maybe admimstered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly. Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions. The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic sol ents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxy-methylcellulose, and or polyvinyl pyrrolidone (PNP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coating. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or agnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

Effective Amounts The Oxalobacter compositions described above are preferably administered to a mammal (e.g., human) in an effective amount, that is, an amount capable of producing a desirable result in a treated mammal. The composition of the invention can be admimstered to a patient either by themselves, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s). In treating a patient exhibiting a disorder of interest, a therapeutically effective amount of an agent or agents such as these is administered. A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Such a therapeutically effective amount can be determined as described below. Toxicity and therapeutic efficacy of the compositions utilized in methods of the invention can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD₅₀ (the dose lethal to 50% of the population) and the ED₅o (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Those compositions that exhibit large therapeutic indices are preferred. While those that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects. The dosage of preferred compositions lies preferably within a range that includes an ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. As is well known in the medical and veterinary arts, dosage for any one animal depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently. It is expected that an appropriate dosage for oral administration of encapsulated Oxalobacter cells would be in the range of about 1 x 10⁷ cells/kg body weight. The methods and compositions of the invention are used to treat patients, preferably humans, with high urinary or serum oxalate levels or hyperoxaluria or who are at risk of high urinary or serum oxalate levels or hyperoxaluria. For example, patients who can be treated by the administration of the compositions described herein include those who have or have had urinary calculi or kidney stones, those who have renal deficiency due to elevated oxalate levels, those who are on diets containing large amounts of oxalate, those who have ileal disease, ileal resection or jejeunoileal bypass, those who have biliary or pancreatic disease and those with a family history of calculi. Additionally, patients with cardiomyopathy, cardiac conductance disorders, cystic fibrosis, Crohn's disease, renal failure, vulvodynia and depleted colonies of intestinal Oxalobacter formigenes. Other renal diseases that are suitable for treatment using the compositions and methods of the invention, include but not limited to: chronic renal diseases (nephropathy, renal failure, nephritis) and acute renal diseases. Specific examples include acute renal failure, chronic renal failure, glomerulonephritis, nephrotic syndrome, systemic lupus erythematosus, glomerular diseases accompanying hepatic disease, diabetic nephropathy, tubulointerstitial nephritis, renal vascular disorder, hypertensive renal disorder, nephro-urinary calculus, urinary tract infection, occlusive nephropathy, cystic renal disease, nephro-urinary tumor, hereditary renal disease, kidney transplant, complication by kidney transplant, and drug-induced renal disorder. Examples of occlusive lesion are lower limb ischemia, acute arterial occlusion, chronic arterial occlusion, arteriosclerosis obliterans, arterial embolism, arterial thrombosis, Buerger's disease, venous occlusion, venous thrombosis, thrombotic phlebitis, angiodysplasia, vascular damage, coronary occlusion, coronary stenosis, pulmonary embolism, and arterial occlusion of organs. The exact formulation, roxite of administration and dosage can be chosen by the individual physician in view of tfcie patient's condition. (See e.g. Fingl et al., in The Pharmacological Basis of Thera eutics, 1975, Ch. 1 p. 1). It should be noted that the attending physician would know liow and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the renal disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

Identification and Quantitation oj ^' Lysate/Enzyme Preparations In another preferred embodiment, a method of treating a patient suffering from renal disease comprises administering to the patient a composition comprising oxalate substrate specific Oxalobacter lysates and Oxalobacter enzymes, wherein administration of the composition increases the rate of oxalate transport from a parenteral site to the intestinal lumen. Preferably, the Oxalobacter lysates and Oxalobacter enzymes are in a ratio of 1:1 (v/v) up to a ratio of 1 : 50 (v/v). In accordance with the indention, these ratios can be manipulated according to the patient's response to the treatment. Therefore, in some patients the Oxalobacter lysates and Oxalobacter enzymes are in a ratio of 5:1 (v/v) up to a ratio of 50:1 (v/v). In a preferred embodiment, the method further provides administration of oxalate- degrading enzymes, such as, formyl-CoA transferase and oxalyl CoA decarboxylase. The enzymes can be administered before, during and/or after administration of the composition comprising Oxalobacter lysates and Oxalobacter enzymes. In a preferred embodiment, the composition comprising Oxalobacter lysates and Oxalobacter enzymes provide an oxalate degrading capacity of about 50 to about 900mg oxalate/day. Each dose comprises about 1. Omg to about 20 mg of active enzyme component with an oxalate degrading capacity between about 0.1 μmole to about 100.0 μmole oxalate/ min/mg protein. Methods for identification and/or quantitation of lysate/enzyme preparations can be obtained by methods well-known in the art. These include, without limitation immunoassays, gels, spectrometry, and the like. Preparation and u.se of the lysate/enzymes are described in detail in the Examples which follow. Methods for mass-scale and rapid quantitation are described in U.S. Patent No.: 6,852,544, incorporated herein by reference. The described methods are useful in the analysis of complex mixtures of proteins, for example, those containing 5 or more distinct proteins or protein functions. U.S. Patent No.: 6,852,544 provides analytical reagents and mass spectrometry-based methods using these reagents for the rapid, and quantitative analysis of proteins or protein function in mixtures of proteins. The analytical method can be used for qualitative and particularly for quantitative analysis of global protein expression profiles in cells and tissues. The method can also be employed to screen for and identify proteins whose expression levels in cells, tissue or biological fluids are affected by a stimulus (e.g., administration of the compositions described herein), by a change in environment (e.g., nutrient level, temperature, passage of time) or by a change in condition or cell state (e.g., disease state, malignancy, site-directed mutation, gene knockouts) of the cell, tissue or organism from which the sample originated. The proteins identified in such a screen can function as molecules for the changed state. For example, comparisons of protein expression profiles of nomial and renal disease cells can result in the identification of proteins whose presence or absence is characteristic and diagnostic of the renal disease. These methods can be employed to screen for changes in the expression or state of enzymatic activity of specific proteins and are useful for diagnosing enzyme-based diseases and for investigating complex regulatory networks in cells. The methods are also used to implement a variety of clinical and diagnostic analyses to detect the presence, absence, deficiency or excess of a given protein or protein function in a biological fluid (e.g., blood), or in cells or tissue. Typically, preparation involves fractionation of the sample and collection of fractions determined to contain the molecules. Methods of pre-fractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity chromatography, sequential extraction, gel electrophoresis and liquid chromatography. The analytes also may be modified prior to detection. These methods are useful to simplify the sample for further analysis. For example, it can be useful to retnove high abundance proteins, before analysis. In one embodiment, a sample, i.e. Oxalobacter lysate an<l/or enzyme preparation, can be pre-fractionated according to size of proteins in a sample using size exclusion chromatography. For a biological sample wherein the amount of sample available is small, preferably a size selection spin column is used. In general, the first fraction that is eluted from the column ("fraction 1") has the highest percentage of high molecular weight proteins; fraction 2 has a lower percentage of high molecular weight proteins; fraction 3 has even a lower percentage of high molecular weight proteins; fraction 4 hias the lowest amount of large proteins; and so on. Each fraction can then be analyzed by immxmoassays, gas phase ion spectrometry, and the like, for the detection of active fractions. In another embodiment, a sample can be pre-fractionated by anion exchange chromatography. Anion exchange chromatography allows pre-fractionation of the proteins in a sample roughly according to their charge characteristics. For example, a Q anion-exchange resin can be used (e.g., Q HyperD F, Biosepra), and a sample can be sequentially eluted with eluants having different pH's. Anion exchange chromatography allows separation of molecules in a sample that are more negatively charged from other types of molecules. Proteins that are eluted with an eluant having a high pH is likely to be weakly negatively charged, and a fraction that is eluted with an eluant having a low pH is likely to be strongly negatively charged. Thus, in addition to reducing complexity of a sample, anion exchange chromatography separates proteins according to their binding characteristics. In yet another embodiment, a sample can be pre-fractionated by heparin chromatography. Heparin chromatography allows pre-fractiona-tion of the molecules in a sample also on the basis of affinity interaction with heparin and charge characteristics. Heparin, a sulfated mucopolysaccharide, will bind molecules with positively charged moieties and a sample can be sequentially eluted with eluants having different pH's or salt concentrations. Molecules eluted with an eluant having a low pΗ are more likely to be weakly positively charged. Molecules eluted with an eluant havnng a high pH are more likely to be strongly positively charged. Thus, heparin chromatograpt y also reduces the complexity of a sample and separates molecules according to their binding characteristics. In yet another embodiment, a sample can be pre-fractionated by isolating proteins that have a specific characteristic, e.g. are glycosylated. For example, a blood, or serum sample can be fractionated by passing the sample over a lectin chromatography column (which has a high affinity for sugars). Glycosylated proteins will bind to the lectin column and non- glycosylated proteins will pass through the flow through. Glycosylated proteins are then eluted from the lectin column with an eluant containing a sugar, e.g., N-acetyl-glucosamine and are available for further analysis. Thus there are many ways to reduce the complexity of a sample based on the binding properties of the proteins in the sample, or the characteristics of the proteins in the sample. In yet another embodiment, a sample can be fractionated using a sequential extraction protocol. In sequential extraction, a sample is exposed to a series of adsorbents to extract different types of molecules from a sample. For example, a sample is applied to a first adsorbent to extract certain proteins, and an eluant containing non-adsorbent proteins (i.e., proteins that did not bind to the first adsorbent) is collected. Then, the fraction is exposed to a second adsorbent. This further extracts various proteins from the fraction. This second fraction is then exposed to a third adsorbent, and so on. Any suitable materials and methods can be used to perform sequential extraction of a sample. For example, a series of spin columns comprising different adsorbents can be used. In another example, a multi-well plate/system comprising different adsorbents at its bottom can be used. In another example, sequential extraction can be performed on a probe adapted for use in a gas phase ion spectrometer, wherein the probe surface comprises adsorbents for binding molecules. In this embodiment, the sample is applied to a first adsorbent on the probe, which is subsequently washed with an eluant. Molecules that do not bind to the first adsorbent are removed with an eluant. The molecules that are in the fraction can be applied to a second adsorbent on the probe, and so forth. The advantage of performing sequential extraction on a gas phase ion spectrometer probe is that molecules that bind to various adsorbents at every stage of the sequential extraction protocol can be analyzed directly using a gas phase ion spectrometer. In yet another embodiment, molecules in a sample can be separated by high- resolution electrophoresis, e.g., one or two-dimensional gel electrophoresis. A fraction containing an enzyme can be isolated and further analyzed by gas phase ion spectrometry. Preferably, two-dimensional gel electrophoresis is used to generate two-dimensional array of spots of molecules, including one or more molecules. See, e.g., Jungblut and Thiede, Mass Spectr. Rev. 16:145-162 (1997). The two-dimensional gel electrophoresis can be performed using methods known in the art. See, e.g., Deutscher ed., Methods In Enzymology vol. 182. Typically, molecules in a sample are separated by, e.g., isoelectric focusing, during which molecules in a sample are separated in a pH gradient until they reach a spot where their net charge is zero (i.e., isoelectric point). This first separation step results in one-dimensional array of molecules. The molecules in one dimensional array are further separated using a technique generally distinct from that used in the first separation step. For example, in the second dimension, molecules separated by isoelectric focusing are further separated using a polyacrylamide gel, such as polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS- PAGE). SDS-PAGE gel allows further separation based on molecular mass of molecules. Typically, two-dimensional gel electrophoresis can separate chemically different molecules in the molecular mass range from 1000-200,000 Da within complex mixtures. Molecules in the two-dimensional array can be detected ixsing any suitable methods known in the art. For example, molecules in a gel can be labeled or stained (e.g., Coomassie Blue or silver staining). If gel electrophoresis generates spots that correspond to the molecular weight of one or more molecules of the invention, the spot can be further analyzed by densitometric analysis or gas phase ion spectrometry. For example, spots can be excised from the gel and analyzed by gas phase ion spectrometry. Alternatively, the gel containing molecules can be transferred to an inert membrane by applying an electric field. Then a spot on the membrane that approximately corresponds to the molecular weight of, for example, an enzyme can be analyzed by gas phase ion spectrometry. In gas pliase ion spectrometry, the spots can be analyzed using any suitable techniques, such as MAXDI or SELDI. Prior to gas phase ion spectrometry analysis, it may be desirable to cleave molecules in the spot into smaller fragments using cleaving reagents, such a-s proteases (e.g., trypsin). The digestion of molecules into small fragments provides a mass fingerprint of the molecules in the spot, which can be used to determine the identity of molecules if desired. In yet another embodiment, high performance liquid chromatography (HPLC) can be used to separate a mixture of molecules in a sample based on their different physical properties, such as polarity, charge and size. HPLC instruments typically consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Molecules in a sample are separated by injecting an aliquot of the sample onto the column. Different molecules in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase. A fraction that corresponds to the molecular weight and/or physical properties of one or more molecules can be collected. The fraction can then be analyzed by gas phase ion spectrometry to detect molecules. Optionally, a molecule can be modified before analysis to improve its resolution or to determine its identity. For example, the molecules maybe subject to proteolytic digestion before analysis. Any protease can be used. Proteases, such as trypsin, that are likely to cleave the molecules into a discrete number of fragments are particularly useful. The fragments that result from digestion function as a fingerprint for the molecules, thereby enabling their detection indirectly. This is particularly useful where there are molecules with similar molecular masses. Also, proteolytic fragmentation is useful for high molecular weight molecules because smaller molecules are more easily resolved by mass spectrometry. In another example, molecules can be modified to improve detection resolution. For instance, neuraminidase can be used to remove terminal sialic acid residues from glycoproteins to improve binding to an anionic adsorbent and to improve detection resolution. In another example, the molecules can be modified by the attachment of a tag of particular molecular weight that specifically bind to molecular molecules, further distinguishing them. Optionally, after detecting such modified molecules, the identity of the molecules can be further determined by matching the physical and ch-emical characteristics of the modified molecules in a protein database (e.g., SwissProt). After preparation, molecules in a sample are typically captured on a substrate for detection. Traditional substrates include antibody-coated 96-well plates or nitrocellulose membranes that are subsequently probed for the presence of proteins. Preferably, the molecules are identified using immunoassays as described above. However, preferred methods also include the use of biochips. Preferably the biochips are protein biochips for capture and detection of proteins. Many protein biochips are described in the art. These include, for example, protein biochips produced by Packard BioScience Company (Meriden CT), Zyomyx (Hayward, CA) and Phylos (Lexington, MA). In general, protein biochips comprise a substrate having a surface. A capture reagent or adsorbent is attached to the surface of the substrate. Frequently, the surface comprises a plurality of addressable locations, each of which location has the capture reagent bound there. The capture reagent can be a biological molecule, such as a polypeptide or a nucleic acid, which captures other molecules in a specific manner. Alternatively, the capture reagent can be a chromatographic material, such as an anion exchange material or a hydrophilic materiaJ. Examples of such protein biochips are described in the following patents or patent applications: U.S. patent 6,225,047 (Hutchens and Yip, "Use of retentate chromatography to generate difference maps," May 1, 2001), International publication WO 99/51773 (Kuimelis and Wagner, "Addressable protein arrays," October 14, 1999), International publication WO 00/04389 (Wagner et al., "Arrays of protein-capture agents and methods of use thereof," July 27, 2000), International publication WO 00/56934 (Englert et al, "Continuous porous matrix arrays," September 28, 2000). In general, a sample is placed on the active surface of a biochip for a sufficient time to allow binding. Then, unbound molecules are washed from the surface using a suitable eluant. In general, the more stringent the eluant, the more tightly the proteins must be bound to be retained after the wash. The retained protein molecules now can be detected by appropriate means. Analytes captured on the surface of a protein biochip can be detected by any method known in the art. This includes, for example, mass spectrometry, fluorescence, surface plasmon resonance, ellipsometry and atomic force microscopy. Mass spectrometry, and particularly SELDI mass spectrometry, is a useful method for detection of the molecules of this invention. Preferably, a laser desorption time-of-flight mass spectrometer is used in embodiments of the invention. In laser desorption mass spectrometry, a substrate or a probe comprising molecules is introduced into an inlet system. The molecules are desorbed and ionized into the gas phase by laser from the ionization source. The ions generated are collected by an ion optic assembly, and then in a time-of-flight mass analyzer, ions are accelerated through a short high voltage field and let drift into a high vacuum chamber. At the far end of the high vacuum chamber, the accelerated ions strike a sensitive detector surface at a different time. Since the time-of- flight is a function of the mass of the ions, the elapsed time between ion formation and ion detector impact can be used to identify the presence or absence of molecules of specific mass to charge ratio. Matrix-assisted laser desorption/ionization mass spectrometry, or MALDI-.MS, is a method of mass spectrometry that involves the use of an energy absorbing molecule, frequently called a matrix, for desorbing proteins intact from a probe surface. MA LDI is described, for example, in U.S. patent 5,118,937 (Hillenkamp et al.) and U.S. patent 5,045,694 (Beavis and Chait). In MALDI-MS the sample is typically mixed with a matrix material and placed on the surface of an inert probe. Exemplary energy absorbing molecules include cinnamic acid derivatives, sinapinic acid ("SPA"), cyano hydroxy cinnamic acid ("CHCA") and dihydroxybenzoic acid. Other suitable energy absorbing molecules are known to those skilled in this art. The matrix dries, forming crystals that encapsulate the analyte molecules. Then the analyte molecules are detected by laser desorption/ionization mass spectrometry. MALDI-MS is useful for detecting the molecules of this invention if the complexity of a sample has been substantially reduced using the preparation methods described above. Surface-enhanced laser desorption ionization mass spectrometry, or SELDI-MS represents an improvement over MALDI for the fractionation and detection of biomolecules, such as proteins, in complex mixtures. SELDI is a method of mass spectrometry in which biomolecules, such as proteins, are captured on the surface of a protein biochip using capture reagents that are bound there. Typically, non-bound molecules are washed from the probe surface before interrogation. SELDI is described, for example, in: United States Patent 5,719,060 ("Method and Apparatus for Desorption and Ionization of Analytes," Hutchens and Yip, February 17, 1998,) United States Patent 6,225,047 ("Use of Retentate Chromatography to Generate Difference Maps," Hutchens and Yip, May 1, 2001) and Weinberger et al, "Time-of-flight mass spectrometry," in Encyclopedia of Analytical Chemistry, R.A. Meyers, ed., pp 11915-11918 John Wiley & Sons Chichesher, 2000. Molecules on the substrate surface can be desorbed and ionized using gas phase ion spectrometry. Any suitable gas phase ion spectrometers can be used as long as it allows molecules on the substrate to be resolved. Preferably, gas phase ion spectrometers allow quantitation of molecules. In one embodiment, a gas phase ion spectrometer is a mass spectrometer. In a typical mass spectrometer, a substrate or a probe comprising molecules on its surface is introduced into an inlet system of the mass spectrometer. The molecules are then desorbed by a desorption source such as a laser, fast atom bombardment, high energy plasma, electrospray ionization, thermospray ionization, liquid secondary ion MS, field desorption, etc. The generated desorbed, volatilized species consist of preformed ions or neutrals which are ionized as a direct consequence of the desorption event. Generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions. The ions exiting the mass analyzer are detected by a detector. The detector then translates information of the detected ions into mass-to-charge ratios. Detection of the presence of molecules or other substances will typically involve detection of signal intensity. This, in turn, can reflect the quantity and character of molecules bound to the substrate. Any of the components of a mass spectrometer (e.g., a desorption source, a mass analyzer, a detector, etc.) can be combined with other suitable components described herein or others known in the art in embodiments of the invention. In another embodiment, an immunoassay can be used to detect and analyze molecules in a sample. This method comprises: (a) providing an antibody that specifically binds to a molecule of interest; (b) contacting a sample with the antibody; and (c) detecting the presence of a complex of the antibody bound to the molecule in the sample. To prepare an antibody that specifically binds to an unknown molecule, purified molecules or their nucleic acid sequences can be used. Nucleic acid and amino acid sequences for molecules can be obtained by further characterization of these molecules. For example, each marker can be peptide mapped with a number of enzymes (e.g., trypsin, N8 protease, etc.). The molecular weights of digestion fragments from each marker can be used to search the databases, such as SwissProt database, for sequences that will match the molecular weights of digestion fragments generated by various enzymes. Using this method, the nucleic acid and amino acid sequences of other molecules can be identified if these molecules are known proteins in the databases. Alternatively, the proteins can be sequenced using protein ladder sequencing. Protein ladders can be generated by, for example, fragmenting the molecules and subjecting fragments to enzymatic digestion or other methods that sequentially remove a single amino acid from the end of the fragment. Methods of preparing protein ladders are described, for example, in International Publication WO 93/24834 (Chait et al.) and United States Patent 5,792,664 (Chait et al.). The ladder is then analyzed by mass spectrometry. The difference in the masses of the ladder fragments identify the amino acid removed from the end of the molecule. If the molecules are not known proteins in the databases, nucleic acid and amino acid sequences can be determined with knowledge of even a portion of the amino acid sequence of the molecule. For example, degenerate probes can be made based on the Ν-terminal amino acid sequence of, for example, the active molecule in the lysate. These probes can then be used to screen a genomic or cDΝA library created from a sample from which a molecule was initially detected. The positive clones can be identified, amplified, and their recombinant

DΝA sequences can be subcloned using techniques which are well known. See, e.g., Current Protocols for Molecular Biology (Ausubel et al., Green Publishing Assoc. and Wiley- Interscience 1989) and Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Cold Spring Harbor Laboratory, ΝY 2001). Using the purified molecules or their nucleic acid sequences, antibodies that specifically bind to them can be prepared using any suitable methods known in the art. See, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniques include, but are not limited to, antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)). After the antibody is provided, enzymes or other active molecules in a lysate preparation can be detected and/or quantified using any of suitable immunological binding assays known in the art (see, e.g., U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay. These methods are also described in, e.g., Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991); and Harlow & Lane, supra. Optionally, the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample. Examples of solid supports include glass or plastic in the form of, e.g., a microtiter plate, a stick, a bead, or a microbead.

Antibodies can also be attached to a probe substrate or ProteinChip® array described above. The sample is preferably a lysate of Oxalobacter. The sample can be diluted with a suitable eluant before contacting the sample to the antibody. After incubating the sample with antibodies, the mixture is washed and the antibody- antigen complex formed can be detected. This can be accomplished by incubating the washed mixture with a detection reagent. This detection reagent may be, e.g., a second antibody which is labeled with a detectable label. Exemplary detectable labels include magnetic beads (e.g., DYNABEADS™), fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads. Alternatively, the active molecules in an Oxalobacter lysate can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound active molecule-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the molecule is incubated simultaneously with the mixture. Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, molecule, volume of solution, concentrations and the like. Usually the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C. Immunoassays can be used to determine presence or absence of a protein in a sample as well as the quantity of a protein in a sample. First, a test amount of a protein in a sample can be detected using the immunoassay methods described above. If a protein is present in the sample, it will form an antibody-protein complex with an antibody that specifically binds the protein under suitable incubation conditions described above. The amount of an antibody-protein complex can be determined by comparing to a standard. A standard can be, e.g., a known compound or another protein known to be present in a sample. As noted above, the test amount of protein need not be measured in absolute units, as long as the unit of measurement can be compared to a control. The methods for detecting these molecules in a sample have many applications. For example, one or more molecules can be measured to aid in the diagnosis of renal disorders. In another example, the methods for detection of the molecules can be used to monitor responses of a subject to treatment. In another example, the methods for detecting molecules can be used to assay for and to identify compounds that modulate expression of these molecules in vivo or in vitro. Data generated by desorption and detection of molecules can be analyzed using any suitable means. In one embodiment, data is analyzed with the use of a programmable digital computer. The computer program generally contains a readable medium that stores codes. Certain code can be devoted to memory that includes the location of each feature on a probe, the identity of the adsorbent at that feature and the elution conditions used to wash the adsorbent. The computer also contains code that receives as input, data on the strength of the signal at various molecular masses received from a particular addressable location on the probe. This data can indicate the number of molecules detected, including the strength of the signal generated by each marker. Data analysis can include the steps of determining signal strength (e.g., height of peaks) of a marker detected and removing "outliers" (data deviating from a predetermined statistical distribution). The observed peaks can be normalized, a process whereby the height of each peak relative to some reference is calculated. For example, a reference can be background noise generated by instrument and chemicals (e.g., energy absorbing molecule) which is set as zero in the scale. Then the signal strength detected for each marker or other biomolecules can be displayed in the form of relative intensities in the scale desired (e.g., 100). Alternatively, a standard (e.g., a serum protein) may be admitted with the sample so that a peak from the standard can be used as a reference to calculate relative intensities of the signals observed for each marker or other molecules detected. The computer can transform the resulting data into various formats for displaying. In one format, referred to as "spectrum view or retentate map," a standard spectral view can be displayed, wherein the view depicts the quantity of marker reaching the detector at each particular molecular weight. In another format, referred to as "peak map," only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling molecules with nearly identical molecular weights to be more easily seen. In yet another format, referred to as "gel view," each mass from the peak view can be converted into a grayscale image based on the height of each peak, resulting in an appearance similar to bands on electrophoretic gels. In yet another format, referred to as "3-D overlays," several spectra can be overlaid to study subtle changes in relative peak heights. In yet another format, referred to as "difference map view," two or more spectra can be compared, conveniently highlighting unique molecules and molecules which are up- or down-regulated between samples. Protein profiles (spectra) from any two samples may be compared visually. In yet another format, Spotfire Scatter Plot can be used, wherein molecules that are detected are plotted as a dot in a plot, wherein one axis of the plot represents the apparent molecular mass of the molecules detected and another axis represents the signal intensity of molecules detected. For each sample, molecules that are detected and the amount of molecules present in the sample can be saved in a computer readable medium. These data can then be compared to a control (e.g., a profile or quantity of molecules detected in control, e.g., normal, healthy subjects in whom renal disease is undetectable). The following examples are offered by way of illustration, not by way of limitation. While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more of the features of the previously described embodiments can be combined in any manner with one or more features of any other embodiments in the present invention. Furthermore, many variations of the invention will become apparent to those skilled in the art upon review of the specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents. All publications and patent documents cited in this application are incorporated by reference in pertinent part for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is "prior art" to their invention.

EXAMPLES Materials and Methods Growth of Oxalobacter and preparation of Oxalobacter-derived products: Growth of bacterial strains was conducted in large fermenter vessels. The strains selected to represent the diversity within the genus Oxalobacter include three from Group I: strain OxB, the type strain; strain OxWR isolated from a wild rat; and strain HC-1 from a human fecal sample. The two strains from Group II are: strain OxCR from a laboratory rat and strain BA-1 from a human fecal sample. The various lysate preparations made from each of these strains used at three different concentrations in separate series of flux experiments (final protein concentration 0.54 mg/ml of mucosal bathing solution was used in experiments). (i) Oxalobacter formigenes, is grown anaerobically under a CO₂ atmosphere at 37° C in medium (pH=7.0) which contains trace metals and the following compounds in gm L: 13.4 sodium oxalate, 4.0 sodium carbonate, 1.0 yeast extract, 0.82 sodium acetate, 0.5 ammonium sulfate, 0.5 cystein hydrochloride, 0.25 potassium phosphate dibasic, 0.25 magnesium sulfate, and 0.001 resazurin. In order to produce the supplemental supplies, the bacteria are grown in either 100 liter or 15 liter fermenter vessels and harvested using a continuous flow centrifuge. The oxalate degradative capacity of the cell preparation is determined as described previously (Allison, M. J. et al., Arch Microbiol 141: 1-7, 1985.) in terms of ¹ CO₂ produced from ¹⁴C-oxalate during incubation in rubber-stoppered test tubes. (ii) Cell lysates of Oxalobacter strains are obtained by rupturing the cells using a French Press. Briefly, the frozen cell paste is re-suspended in the lysis buffer ( 0.1M potassium phosphate buffer, pH 6.8, containing lOmM MgCl₂, ImM Thiamine diphosphate and ImM dithiothreitol). Cell lysis is performed in the French-Press at a pressure of about 1000 bar (2-3 passes). To ensure inhibition of proteolytic activity 2 mM aprotinin is added to the cell suspension. The lysed cells are centrifuged at 15,000 g for 20 minutes to remove cell debris. The lysate is further clarified by ultrafiltration through 0.45 μ filter. This final lysate preparation is immediately stored at -80°C. In addition, an aliquot is subjected to lyopholization. Each batch of the lysate is tested for protein concentration, protein profile by SDS-PAGE chromatography and for its oxalate degrading activity. Enzyme activity is measured before the lysate is frozen and again when it is thawed out for testing in the flux experiments. (iii) Determination of oxalate degrading activity of the O. formigenes lysate: The determination of oxalate degrading capacity is performed by the method of Baetz et al, (Baetz, A. L., and M. J. Allison. JBacteriol 171: 2605-8, 1989.). This assay takes advantage of the degradation of oxalate to formate and carbon dioxide by the enzyme system. The formate produced is then measured in terms of conversion of NAD to NADH by formate dehydrogenase. The reaction mixture, contains KH PO₄ (pH 6.1), 66 mM; TPP, 0.06 mM; Magnesium chloride, 6.6 mM; potassium oxalate, 8.3mM; oxalyl-CoA, 0.5 mM; NAD, 0.83mM; formate dehydrogenase, 0.25 LU. The reaction is initiated by addition of

Oxalobacter lysate as the source of formyl-CoA transferase and oxalyl-CoA decarboxylase. Initial rates of the reaction are recorded and calculated by the increase in absorbance at 340 nm using the extinction coefficient for NAD.

Enhancement of large-scale production of Oxalobacter: Initial trials are to be conducted with selected acetogens, and Oxalobacter formigenes, strain HC-1. The latter is a rapidly growing strain that was isolated from human feces and which is a promising strain for product development. Measurements are made of growth of individual pure cultures and co-cultures (acetogen with Oxalobacter) with varied levels of oxalate in the culture media. Measurements of oxalate and of end products (formate and acetate) are made from samples taken during and at the end of growth. Growth is measured as optical density (calibrated to cell dry weight) in test tube cultures grown under anaerobic conditions. Oxalate, formate and acetate are measured by gas chromatography of their butyl esters. Co-cultures with selected acetogens are then carried out in a one liter bench top fermenter that is equipped with a pH stat system so that oxalic acid can automatically be fed to the culture in an amount that is related to the rate of oxalate degradation. Based on co-culture results, the acetogen that is most promising as a formate remover is selected for further tests. This acetogen is grown in a large fermenter (12 to 100 liters are available) and cells are harvested in late log phase to retain maximum metabolic capacity. These cells are placed as concentrated cell suspensions within dialysis bags that are placed inside culture vessels with growing Oxalobacter cells. Formate is consumed by the acetogen and the Oxalobacter culture, freed of formate inhibition grows to produce a much larger crop of cells per unit volume of culture medium. Additional substrate, as oxalic acid, is fed in response to the increase in pH. An alternative to this experimental design is to have parallel cultures of Oxalobacter and acetogen growing in (e.g. 20 liter carboys) and to pump the acetogen culture through a dialysis bag, or other exchange system, suspended in the Oxalobacter culture. An "Ecologen" apparatus (New Brunswick Instrument Co.), which provides for parallel cultures in contact with each other but with cells separated by millipore filters is also available for these studies. Other methods for transfer of formate to the formate user, such as flow through hollow fiber systems are examined as will using immobilized cells of the fom ate user

Preparation of capsules containing supplemental supplies of Oxalobacter: Briefly, a cell lysate is prepared from one of the strains of Oxalobacter. The choice of strain is one which produces optimal colonic oxalate secretion and the lysate with known oxalate degrading capacity (measured in an in vitro assay) are placed in the capsules with oxalyl CoA, TPP and MgCl₂ as co-factors. The minicapsules (size 9) especially designed for rat studies (Torpac Inc., NJ. Torpac Inc., also sells a capsule filling and feeding device that has been successfully used for prior studies) is coated with Eudragit L 100-55 (Huls America Inc., NJ). Eudragit L 100-55 provides an enteric coating to the capsules that safely takes the supplement through the highly acidic gastric contents. It was demonstrated, in in vitro studies, that Eudragit coated capsules showed no dissolution at 37° C at pH < 6.0. The capsules provide an oxalate degrading capacity of approximately 200-500mg oxalate /day. Each capsule contains about 1.5 - 2 mg of active enzyme component with an oxalate degrading capacity between 0.8 - 1.0 μmole oxalate/ min/mg protein. The capsules are placed at the end of an intragastic needle and administered to the rats by intragastric intubation at the beginning and the end of each day, for a 7-day period.

Oxalate Flux Studies: Oxalate flux measurements using in vitro intestinal tissue preparations are routine in applicant's laboratory. Briefly, the distal colon is removed from euthanized rats. The colonic segments are rinsed in a standard buffered saline and the sub-mucosal connective tissues and muscle layers are removed using blunt dissection. Sheets of colonic tissue are mounted in Ussing chambers with an exposed area of 0.64 cm². Standard saline solutions bathe either face of the tissue and these are maintained at 37° C and circulated by bubbling with a gas mixture, 95% O₂ / 5% CO₂. Transepithelial fluxes of ¹⁴C-oxalate (Amersham Corp., Arlington Heights, IL, with a specific activity of 27 mCi/mmol)) are measured under short-circuit conditions with an automatic voltage clamping device, (VCC600, Physiologic Instruments, San Diego, CA) as described previously (Freel, R. W., M. Hatch, D. L. Earnest, and A. M. Goldner. Oxalate transport across the isolated rat colon. Biochim Biophys Ada 600: 838-43, 1980; Hatch, M., R. W. Freel, andN. D. Vaziri. Am Soc Nephrol 5: 1339-43, 1994).

Evaluation of Potential Secretagogue Effects of Oxalobacter Cell Lysates In Vitro: To establish the effects of cell lysate preparations on oxalate transport in vitro, a two period protocol is employed. Since these preparations can degrade oxalate in the Ussing chamber environment each one is heat-treated before it is used in the flux study. Plunging a tube containing the lysate preparation into boiling water for 1 minute eradicates this degradative activity. In the first period, three measurements of oxalate flux are determined at 15-minute intervals. After a 15-minute equilibration period, a second sampling period is initiated comprising three 15 minute sampling intervals. The unidirectional fluxes for the control (Per I) and the experimental (Per II) are obtained by averaging the three sampling intervals comprising that period. Tissue electrical parameters are similarly treated to obtain control and experimental values.

Removal of Kidneys and Evaluation of Renal Damage: Animals are euthanized with an intraperitoneal injection of sodium pentobarbital. The kidneys are removed and fixed in 10% neutral buffered formalin, trimmed, processed, and embedded in paraffin. Two sections from each kidney are stained with hematoxylin and eosin and examined under polarized light. The presence of CaOx crystals are scored on a basis of 0-4+ . The rest of the kidney tissue is saved for the determination of other indicators of renal injury as follows:

Determination of lipid peroxides. Urinary lipid peroxides as malondialdehyde coupled with thiobarbituric acid will be assayed by HPLC using a Water's Φ-Bondapak C18 column, eluting with phosphate- methanol (65%-35%) mobile phase. The eluted malondialdehyde complex which has a retention time of 8 minutes, is measured at 532 nm. Tetraethoxypropane is used as a standard. A portion of the kidney tissue is used to determine the products of lipid peroxidation. The kidney tissues for assay are rapidly excised, decapsulated, blotted, and minced with fine scissors. The samples are homogenized in 10 ml of cold 154 mM potassium chloride using a tissue homogenizer. Duplicate aliquots of 0.1 of each homogenate are pipetted into pyrex tubes containing 3 ml of cold phosphoric acid, 1 ml of thiobarbituric acid solution and placed for 45 minutes in a boiling water bath. The tubes are cooled to room temperature and the pink color formed extracted with butanol and measured by colorimetry using tetraethoxypropane as a standard.

Enzyme determinations. Centrifuged urine supernatants are dialyzed for three hours at 4°C against deionized water for determination of the following enzymes. Alanine aminopeptidase, a marker of tubular brush border, will be measured at 410 run and 37°C by monitoring the increase of absorbance due to release of 4-nitroaniline catalyzed by alanine aminopeptidase during 3 minute intervals. N-acetyl-glucosaminidase (NAG), a lysosomal marker, is assayed by colorimetry under optimal reaction conditions at 405 nm using 4-nitrophenyl-glycosides as substrate. Lysozyme, after purification with a sep-pak C18 cartridge, is quantified by HPLC on a HS-5 C18 (0.4 x 12.5 cm) column. Samples for lysozyme are analyzed by elution for 25 minutes at a flow rate of 1.5 ml/min with a linear gradient from 45% to 65% acetonitrile with the addition of 0.1 % trifluoracetic acid. Chromatograms are recorded by monitoring absorbance at 220 nm using a LC 85 UN detector (Perkin Elmer) and data processed with L- Cl-100 integrator. Isocitrate dehydrogenase is measured by monitoring the rate of absorbance increase at 340 nm as ΝADP is reduced. Lactate dehydrogenase is analyzed on the spectrophotometer by measuring the rate of decrease of absorbance of ΝADH at 340 nm using pyruvate as substrate.

Urine and Plasma Oxalate Determination: The measurement of oxalate in urine and plasma specimens is routine in our laboratory. The enzymatic, sensitive, and specific assay procedure currently being used was developed by Hatch et al. (Hatch, M. Spectrophotometric determination of oxalate in whole blood. Clin. Chim. Acta 193: 199, 1990; Hatch, M., E. Bourke, and J. Costello. New enzymatic method for serum oxalate determination. Clin. Chem. 23: 76-80, 1977.) and it has been employed to determine oxalate concentrations in numerous studies (using humans and animals). Calcium and creatinine is determined in the urine and plasma samples using the Sigma kit assays #587A and #555A, respectively (Sigma Chemical Co., St. Louis, MO).

Procedure for the Colonization of Rats with Oxalobacter sp. The procedure for colonizing laboratory rats has been described (Cornelius, J. G., M. J. Allison, S. Thamilselvan, and A. B. Peck. Colonization with Oxalobacter formigenes for the regulation of hyperoxaluria. Urolithiasis 2000, edited by A. L. Rodgers, B. E. Hibbert, B. Hess, S. R. Khan and G. M. Preminger. University of Capetown, 2000, p. 185.). Briefly, rats are maintained on a diet containing 0.5% Ca²⁺ / 1.5% oxalate for a period of 8 days before gavage with the live Oxalobacter culture. On days 8 and 9, 1.5 ml of culture medium containing 1-2 x 10⁸ colony forming units of O. formigenes, strain OxWR (wild rat strain of Oxaobacter), is administered to each rat by gavage. DNA is extracted from fresh fecal samples collected at both one and two week intervals after gavage and tested for the presence of Oxalobacter using the XEntrj_x™ O. formigenes Monitor.

Preparation of Rat Chow Diets The standard rat chow that was routinely provided for the Sprague Dawley rats included in all of the studies of oxalate transport over the past 10 years is Purina 5001 and since it is low in oxalate content, it is not suitable for initiating colonization. For the purpose of the colonization procedure, a powdered diet containing 0.5% Ca²⁺ (Product # TD 89222, Harlan-Teklad, Madison, WI) was supplemented with ammonium oxalate (1.5 gram per 100 gram of powder) that was thoroughly mixed into batches before wetting the mixture and rolling it into balls. The food balls were dried at room temperature before they were placed in the rat cages. Throughout the text, for convenience, the composition of a particular diet is referred to by way of the gram amount of oxalate or Ca²⁺ present in that diet per 100 gm of chow i.e. on a percent basis. The low Ca²⁺ diet contained 0.01% Ca²⁺ (Product # TD 99354) and the high Ca²⁺ diet contained 1.2% Ca²⁺ (Product # TD 99355). The oxalate content of each diet, other than the one used to initiate colonization, was 0.5% oxalate. The oxalate content of the diet was altered by simply quantitatively reducing the amount of oxalate salt added to the powder.

Verification of Colonization by Oxalobacter sp. A fresh sample of fecal material taken from the distal colon, at the time this segment was removed for the flux experiment, was snap-frozen in liquid N₂ and shipped to Dr. Sidhu at Ixion. Samples were determined to be Oxα/obαcter-positive or negative by using XEntrj_x™ O. formigenes Monitor.

Flux Studies Male Sprague Dawley rats (285 - 325 g), which were either not colonized or colonized with Oxalobacter sp., were used in the following studies. The rats had free access to drinking water and food at all times. Flux experiments were conducted using distal colonic tissues removed from various groups of rats undergoing different treatments regimens. All of the rats were euthanized by an intraperitoneal injection of sodium pentobarbital (150 mg/kg) prior to removing the tissues. As described previously (Hatch, M., R. W. Freel, and N. D. Vaziri. J. m. Soc. Nephrol 5: 1339-43, 1994.), flat sheets of intestinal tissue were mounted in modified Ussing chambers with an exposed tissue area of 0.64 cm² and bathed on both sides by 10 ml of standard saline solution at 37° C which was circulated by bubbling with a gas mixture, 95% O₂ / 5% CO₂. ¹⁴C-oxalate was obtained from New England Nuclear (Boston, Massachusetts) and all other reagents were purchased from Sigma Chemical Co. (St Louis, Missouri). Transepithelial fluxes of ¹⁴C-oxalate were measured under short-circuit conditions as described previously (Hatch, M., R. W. Freel, and N. D. Vaziri. J. Am. Soc. Nephrol. 5: 1339-43, 1994.). The magnitude and direction of the net flux of oxalate (j^ ) was determined by calculating the difference between two measured unidirectional fluxes (mucosal to serosal, J°* and serosal to mucosal, J°*). The experimental design consisted of a 45 min. period (Period I) during which time fluxes and electrical parameters were measured at 15 min intervals. Tissue conductance (Gχ₅ mS-cm^"2) was calculated as the ratio of the open-circuit potential (mV) to the short-circuit current (I_sc, μA-cm^" ). hi the series involving addition of select bacterial preparations to the in vitro tissue preparation, a similar flux period (Period II) followed Period I after a 15 min equilibration interval. Statistical analysis of the data derived from these experiments was performed by using either an ANOVA for the comparison of multiple means and Duncan's multiple range test or paired and unpaired t-tests for the comparison of two means. Results are expressed as the mean ± 1 SEM. Differences are considered significant if p < 0.05.

Example 1 - Enhancement of Oxalate Excretion Into the Intestinal Lumen of Vertebrates By O. formigenes or Its Products A major goal underlying studies leading to this invention was to determine if enhanced elimination of oxalate into the colon can be induced where it can then be innocuously degraded by O. formigenes. First, a study was conducted to test if O. formigenes colonization could lower urinary oxalate levels in rats fed ethylene glycol (EG), a system that mimics endogenous hyperoxaluria (Fig. 1). Then, a study was conducted to test the hypothesis that colonization of the large intestine by O. formigenes , or that administration of O. formigenes via the gut, might alter oxalate handling by the intestine (Fig. 3 and Fig. 4). In the experiment shown in Figure 4, rats were colonized or not-colonized from birth by rearing with colonized (gavaged with OxWR) or non-colonized mother. Urinary oxalate was significantly lower in colonized rats (5.3 ± 0.5 μmol/24 hours) vs rats not colonized (8.3 ± 0.8 μmol/24 hours). With the underlying premise that O. formigenes can induce or enhance transepithelial oxalate secretion, these latter studies were designed to compare transepithelial transport of oxalate across tissues removed from animals in CRF treated with bacterial lysates of O. formigenes (Fig. 2). The results confirmed the hypothesis that O. formigenes possesses (a) a strategic ability to optimize substrate availability within the intestinal lumen, (b) a capacity to locally modulate epithelial oxalate transport in order to gain increased survival, and (c) a mechanism that can activate the epithelial oxalate transport system. Results of these studies show that Oxalobacter can locally modulate oxalate transport and enhance intestinal elimination. Thus, the action of luminal O. formigenes can greatly impact urinary oxalate excretion. First, the presence of O. formigenes in the intestines lowered urinary oxalate levels 15-20% but increased to 30% reduction when the rats were also fed enough oxalate in their diet to allow stable colonization of the gut with the O. formigenes (Fig. 1). Second, a lysate/enzyme preparation from O. formigenes stimulated the active transport systems involved in colonic oxalate secretion/excretion in addition to enzymatically degrading oxalate intraluminally (Fig. 2). In this experiment, rats received a simultaneous treatment with 0.75% EG in the drinking water and capsules for 5 days BID. Urinary oxalate was reduced 50% (102 +/- 11 to 57 +/- 8 μmol/24 hrs), and Oxalobacter lysate treatment induced local oxalate secretion in the distal colon. Coupled together, these independent and separate actions of O. formigenes and/or lysate/enzyme preparations can promote an enhanced enteric elimination of oxalate. Considered as a whole, then, the administration of O. formigenes, or products thereof, can be used to enhance the extra-renal elimination of oxalate from the circulatory system via the intestines, thereby reducing the burden of oxalate in the kidney. This extra-renal elimination is actually enhanced when CRF is present. The importance of these observations is paramount when one considers hyperoxaluria and oxalosis in the context of the genetic- based primary hyperoxalurias or patients with end-stage renal disease (CRF). In both cases, the kidneys are unable to properly handle the oxalate burden, thus exacerbating renal injury. Lowering systemic oxalate levels through the use of O. formigenes could prove to be of significant benefit to these particular patients. Example 2 — Effectiveness of the enteric lysate/enzyme preparation The proposed mechanism underlying the effectiveness of the enteric lysate/ enzymes preparation is based upon two actions: First, it locally stimulates the active transport systems involved in colonic oxalate secretion. Second, its intraluminal oxalate-degradative capacity serves to sustain an outwardly directed concentration gradient such that transmural passive movement of oxalate from the blood into the lumen is also enhanced. Together, these independent actions of the Oxalobacter lysate/enzyme preparation can optimally promote enteric elimination of oxalate.

Example 3-Effects of Intraluminal Calcium on Urinary Oxalate Excretion in Colonized Rats. The effectiveness of Oxalobacter in reducing urinary oxalate during dietary maneuvers that would alter luminal Ca²⁺ content was examined. Colonized rats, divided into two groups (n=7 each group) were fed diets with either low ("Lo"; 0.01%) or higli ("Hi";

1.2%) calcium for a 3-week; period. The oxalate content (0.5%) was the same in each diet.

The results of the plasma and urinary measurements of creatinine, oxalate and calcium are shown in Table 1.

Table 1

*An asterisk denotes a significant difference between the two groups.

Although this experimental series was aimed at comparing urinary oxalate excretion between the two groups, flux studies were also conducted on the distal colonic segments removed from these animals. Plasma oxalate and calcium concentrations were maintained within normal limits in both groups. There were significant differences between the groups in both urinary oxalate and calcium excretion while plasma and urinary creatinine were comparable. Mean urinary oxalate excretion was increased 15-fold in the group fed the low Ca²⁺ diet compared to the group fed the high Ca²⁺ diet and clearance of oxalate was also significantly higher in the former group. Inversely, mean urinary calcium excretion was decreased 17-fold in the group fed low Ca²⁺ and clearance of calcium was negligible in this group compared to the group fed high Ca²⁺. Although these directional changes in urinary calcium excretion are easily explained on the basis of dietary manipulation alone, it was also clear that mean urinary oxalate ex retion was significantly affected by the Ca²⁺ content of the diet given the oxalate content (0.5%) was the same in both. It was concluded from the results of urinary oxalate excretion in the rats on the low Ca²⁺ diet that colonization with Oxalobacter does not prevent the gross hyperoxaluria that occurs from the obvious increase in luminal oxalate activity and oxalate absorption. Apparently, the luminal oxalate-degradative abilities of Oxalobacter were overwhelmed h y a diet containing 0.5% oxalate and extremely low Ca²⁺. It is also clear from these results that urinary oxalate excretion is affected more by the Ca^2+" content of the diet than by the presence of Oxalobacter. This conclusion was also supported by the fact that the rats fed the high Ca²⁺ diet, lost colonization on that diet and excreted normal amounts of oxalate compared to rats fed the low Ca²⁺ diet.

Example 4 - Induction of Colonic Oxalate Secretion It was hypothesized that Oxalobacter sp. possess a strategic ability to optimize substrate availability within the intestinal lumen. The effects of select bacterial preparations on epithelial oxalate transport were directly tested in a series of in vitro flux experiments. The following preparations were used: preparations of OxWR (wild rat strain of Oxalobacter); a preparation of washed, whole Oxalobacter cells, a preparation of washed Oxalobacter cell membrane fragments, and a preparation of Oxalobacter cell lysate. Distal colonic tissues removed from rats, not colonized and fed the standard Purina chow 5001 diet, were used in these experiments. As mentioned previously, these tissues characteristically support a net absorptive flux of oxalate. Initial experiments quickly revealed that although Oxalobacter is described as an obligative anaerobe, a significant and rapid degradation of oxalate was noted in the standard aerated (95% O₂/5% CO₂) buffers bathing the mucosal side of the colonic preparation following the addition of the bacterial cells. Heat-treatment, by plunging a tube containing the bacterial cell preparation into boiling water for 5 minutes, eradicated this degradative activity and the experiments were subsequently conducted using whole cells (6 mg wet weight / ml of bathing solution) treated in this way. This was not an issue with the cell membrane fragments which were washed 3 times in the standard buffer prior to adding 6 mg wet weight / ml of buffer bathing the mucosal side of the tissue. The flux results from both series of experiments showed that the addition of whole, heat-treated Oxalobacter cells to the mucosal side of the distal colon caused coordinated changes in both unidirectional fluxes resulting in a 58% increase in net oxalate absorption (n=7 tissue pairs from 3 rats). Some effect on the transport of other major ions is also evident as judged by the significant inhibition in I_sc from 4.33 ± 0.51 to 2.39 ± 0.30 μEq-cm^"2.^¹ (G_τ remained unchanged: 10.34 ± 0.64 mS-cm^"2 in Per I and 10.07 ± 1.02 mS-cm^"2 in Per II). Simultaneous measurement of chloride fluxes across these tissues revealed similar alterations in the movements of this anion following the addition of the bacterial preparation; i.e. a significant reduction in J^cl _sm and a significant increase in J^c _net. A similar conclusion was derived from the flux series examining the effects of Oxalobacter cell membrane fragments. These results show (Figure 5) that there were no significant effects of the membrane fragment preparation on oxalate transport across the distal colon (n=9 tissue pairs from 4 rats). While no changes were seen in GT (Per I: Gχ=

10.27 ± 0.49 mS-cm^"2, Per II :

10.17 ± 0.70 mS-cm^"2) a small but significant reduction in 1 1

I_sc from 4.20 ± 0.43 μEq-cm^" -hr^" to 3.51 ± 0.39 μEq-cm^" -hr^" was observed. Since no changes were observed in chloride fluxes across these tissues the effect on I_sc, can be explained by alterations in the movements of another major ion, other than chloride. It was concluded from these two exp erimental series, using heat-treated whole cells and membrane fragments, it was unlikely that the mechanism whereby oxalate secretion is induced involves some cell wall component of Oxalobacter cells. The following experiment examined the effects of a lysate preparation of Oxalobacter cells on colonic oxalate transport. Oxalate fluxes were measured before and after the addition of the lysate to the solution bathing the mucosal side of colonic tissues. The results (Figure 6) showed that addition of an aliquot of the heat-treated lysate (final protein concentration 0.54 mg/ml of mucosal bathing solution) induced a significant net secretion of oxalate across the distal colon (n=7 tissue pairs from 4 rats). No changes were observed in the absorptive component of the flux and the substantial increase in the secretory component accounts for the reversal in the direction of the net flux of oxalate. The electrical characteristics of the tissue also remained unchanged (Per I: Gx = 12.25 ± 1.42 mS-cm^"2, I_sc = 6.90 ± 0.39 μEq-cm^"2-!*^"1; Per II : G_τ= 11.94 ± 1.34 mS-cm^"2, I_sc = 6.08 ± 0.65 μEq-cm^"2-hr^{" l}). It is also evident from this experiment that the secretagogue effect of the lysate (heat- treated) is a separate action independent of the enzymatic degradation of oxalate. Example 5 - The Oxalate-induced Chronic Renal Failure (CRF) Rat Model The aim of the study was to determine the effectiveness of an oxalate-degrading enzyme supplementation therapy on reducing urinary oxalate in renal failure associated with chronic hyperoxaluria. The interest in ^"using this particular model was two-fold: First, the distal colon supports a net secretory fltrx of oxalate that is induced by CRF. Second, persistent hyperoxaluria, due to an endogenous overproduction of oxalate is a feature of this animal model. It was determined that 4 weeks of treating unilateral nephrectomized rats with 0.75% ethylene glycol resulted in a two-fold increase in plasma creatinine. Since results with the 5/6 nephrectomized rat showed that colonic oxalate secretion is initiated and sustained at this level of renal function, the characteristics of oxalate transport in the oxalate-induced CRF rat model were examined. At the time the flux studies were conducted, plasma creatinine was increased about two-fold in ethylene glycol-treated (CRF) rats (0.100 ± 0.006 mM, n = 7) compared to both the non-treated, unilateral nephrectomized (UN) group (0.045 ± 0.O02 mM, n = 8) and a group of normal, healthy controls with both kidneys intact (0.042 ± 0.O03 mM, n = 5). Mean urinary oxalate excretion in the CRF group was significantly greater

(144.4 ± 6.12 μmol/24 hr) than in either the UN group (10.2 ± 1.21 μmol/24 hr) or in normal controls (12.4 ± 1.40 μmol/24 hr). The results of the transport studies (Figure 7) showed that, similar to normal healthy control rats with both kidneys intact, the distal segment of the UN rats supports a significant basal net absorptive flux of oxalate. hi contrast, a reversal in the direction of the net oxalate flux was evident in the CRF rats treated with ethylene glycol and this net secretion of oxalate occurred by way of significant alterations in both of the unidirectional fluxes. Neither I_sc (Control = 4.09 ± 0.05; UN = 4.43 ± 0.37; CRF = 5.30 ± 0.43 μEq-cm^"2-^¹) nor G_τ (Control = 9.86 ± 0.74; UN = 9.80 ± 0.90; CRF = 11.07 ± 0.55 mS-cm^"2) were different among the three groups, n = 7 tissue pairs in each group.

Example 6 - Enzyme supplementation therapy in CRF rats For the purpose of the study examining the effectiveness of an oxalate-degrading enzyme supplementation therapy on reducing urinary oxalate excretion in oxalate-induced CRF rats, unilateral nephrectomized rats were given 0.75% ethylene glycol in their drinking water after a recovery period of one week. Half of the rats were administered enteric-co ated capsules containing Oxalobacter degradative enzymes (a lyophilized preparation of lysate from OxWR, each capsule contained about 1.5 mg of active enzyme component with an oxalate degrading capacity of about 0.6 to 1.0 μmole oxalate/ min/mg protein) twice per day, for a period of 5 days starting on the first day the ethylene glycol regimen began. The other group that served as controls were given empty enteric-coated capsules at similar times. At the end of the study period, urine was collected for oxalate measurements and tissues were removed from all of these rats for oxalate flux studies. A comparison of mean urinary oxalate excretion by the two groups revealed a significant difference. The rats receiving the encapsulated Oxalobacter enzymes excreted almost 50% less oxalate (57.6 ± 7. 9 μmol/24 hours, n=6) than those receiving placebo capsules (102.7 ± 11.42 μmol/24 hours, n=6). This result alone clearly indicates that enzyme supplementation therapy has considerable potential. The results are especially significant because they show that the balance ^"between renal and enteric excretion of endogenously- derived oxalate, in contrast to food oxalate present in the luminal environment, can be manipulated. The oxalate burden in this animal model was derived from ethylene glycol metabolism and the food supplied to these rats was not supplemented with oxalate. Furthermore these results emphasize the important physiological role of intestinal oxalate secretory pathways in shifting the b alance of oxalate excretion between the two routes of elimination. The flux studies revealed more unexpected results (shown in Figure 8). Colonic oxalate transport was significantly altered in the rats receiving the Oxalobacter lysate compared to the placebo-treated rats. A significant colonic secretory flux of oxalate was stimulated in the experimental group as a consequence of the delivery of oxalate degrading enzymes to this intestinal segment. It should be noted here that the fact the placebo-treated rats in this series sustained a small net absorptive flux of oxalate is likely due to the 5 -day duration of the ethylene glycol treatment compared to 30 days in the series presented in Figure 5. After 5 days, plasma creatinine is comparable in both groups in this series but still 40 % lower than the level achieved after 30 days of ethylene glycol administration. This substantial colonic secretion/excretion of oxalate, which is derived from an endogenous source (i.e. metabolism of ethylene glycol), may explain the dramatic reduction in urinary oxalate excretion in this group of rats treated with Oxalobacter lysate/ enzymes. Without wishing to be bound by theory, the mechanism underlying the effectiveness of the enteric lysate/ enzymes preparation is based upon two actions: First, it locally stimulates the active transport systems involved in colonic oxalate secretion. Second, its intraluminal oxalate- degradative capacity serves to sustain an outwardly directed concentration gradient such that transmural passive movement of oxalate from the blood into the lumen is also enhanced. The results obtained in the in vitro experiments using the bacterial lysate (presented above, Figure 6) are consistent with both notions. Together, these independent actions of the Oxalobacter lysate/enzyme preparation can optimally promote enteric elimination of oxalate. In conclusion, it appears prudent to direct the Phase II Specific Aims towards the development of a supplemental lysate/ enzymes therapy for the control of hyperoxaluric conditions.

Other Embodiments It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is: 1. A method for increasing the rate of oxalate trans ort from a parenteral site to an intestinal lumen in a patient suffering from a renal disorder, the method comprising the steps of: administering to the patient a composition comprising oxalate substrate specific Oxalobacter; thereby, increasing the number of oxalate-substrate specific bacteria or amount of oxalate-degrading bacteria enzymes in the gastrointestinal tract in the animal; wherein, the rate of oxalate transport from a parenteral site to the intestinal lumen is increased.

2. The method of claim 1, wherein the Oxalobatcter bacteria colonize the intestines.

3. The method of claim 1, wherein Oxalobacter bacteria locally stimulate active transport systems involved in colonic oxalate secretion.

4. The method of claim 1, wherein the Oxalobacter bacteria stimulate intraluminal oxalate-degradation.

5. The method of claim 4, wherein the oxalate degradation produces an outwardly directed concentration gradient.

6. The method of claim 1, wherein the patient with a renal disorder is a mammal.

7. The method of claim 1, wherein the renal disorder is associated with oxalate levels in the kidneys.

8. The method of claim 1, wherein the method is used to treat a patient with hyperoxaluria.

9. The method of claim 1 , wherein the oxalate-substrate specific bacterium is Oxalobacter formigenes .

10. The method of claim 1 , wherein the step of increasing tTie number of oxalate- substrate specific bacteria or amount of oxalate-degrading bacteria enzymes in the gastrointestinal tract in the animal comprises administering a composition comprising Oxalobacter to the patient.

11. The method of claim 10, wherein the composition further comprises a pharmaceutically acceptable carrier.

12. The method of claim 11, wherein the pharmaceutically acceptable carrier comprises a capsule that is resistant to degradation by gastric acidity.

13. The method of claim 10, wherein the composition is in tablet form.

14. The method of claim 10, wherein the composition is in paste form.

15. The method of claim 10, wherein the composition comprises viable Oxalobacter.

16. The method of claim 17, wherein the composition comprises an active cellular lysate.

17. A method of reducing the amount of time that a subject suffering from renal failure must spend on dialysis, the method comprising the steps of: (A) providing a subject suffering from renal failure; and (B) administering to the subject a composition comprising Oxalobacter bacterium or Oxalobacter enzymes, wherein administration of the composition results in decreased levels of oxalate excretion.

18. A pharmaceutical composition comprising a lysate/enzyme preparation of Oxalobacter bacterium and Oxalobacter enzymes.

19. The pharmaceutical composition of claim 18, wherein the composition comprises a lysate of substrate-specific Oxalobacter bacteria.

20. The pharmaceutical composition of claim 18, wherein the composition comprises oxalate-degrading enzymes.

21. The pharmaceutical composition of claim 18, wherein the oxalate-degrading enzymes are formyl-CoA transferase and oxalyl CoA decarboxylase.

22. The pharmaceutical composition of claim 18, wherein the composition is formulated to prevent degradation in conditions at 37° C at pH < 6.0.

23. The pharmaceutical composition of claim 18, wherein the composition is formulated to provide an oxalate degrading capacity of between about 50 mg to about 900mg oxalate/day.

24. The pharmaceutical composition of claim 18, wherein the composition is formulated to provide an oxalate degrading capacity of between about 200mg to about 500 mg oxalate/day.

25. The phannaceutical composition of claim 18, wherein the composition comprises an oxalate degrading capacity between about 0.1 μmole up to 100 μmole oxalate/min/mg protein.

26. The pharmaceutical composition of claim 18, wherein the composition comprises an oxalate degrading capacity between about 0.1 μmole to about 50.0 μmole oxalate/min/mg protein.

27. The pharmaceutical composition of claim 18, wherein the composition comprises an oxalate degrading capacity between about 0.8 μmole to about 20.0 μmole oxalate/min/mg protein.

28. A method of treating a patient suffering from renal disease comprising: administering to the patient a composition comprising oxalate substrate specific Oxalobacter lysates and Oxalobacter enzymes, wherein administration of the composition increases the rate of oxalate transport from a parenteral site to the intestinal lumen.

29. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme compositions comprise a ratio of 1:1 (v/v).

30. The method of claim 28, wherein the Oxalobacter lysates and Oxalobacter enzymes are in a ratio of 1 :5 (v/v).

31. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme compositions comprise a ratio of 1:10 (v/v).

32. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme compositions comprise a ratio of 1 :50 (v/v).

33. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme compositions comprise a ratio of 5:1 (v/v).

34. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme compositions comprise a ratio of 10:1 (v/v).

35. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme compositions comprise a ratio of 50:1 (v/v).

36. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme compositions are formulated to provide an oxalate degrading capacity of between about 50 mg to about 900mg oxalate/day.

37. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme compositions are formulated to provide an oxalate degrading capacity of between about 200mg to about 500 mg oxalate/day.

38. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme compositions are formulated to provide an oxalate degrading capacity of between about 0.1 μmole up to 100 μmole oxalate/min/mg protein.

39. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme compositions are formulated to provide an oxalate degrading capacity of between about 0.1 μmole up to 100 μmole oxalate/min/mg protein.

40. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme compositions are formulated to provide an oxalate degrading capacity of between about 0.1 μmole to about 50.0 μmole oxalate/min/mg protein.

41. The pharmaceutical composition of claim 28, wherein the composition comprises an oxalate degrading capacity of between about 0.8 μmole to about 20.0 μmole oxalate/min/mg protein.

42. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme composition locally stimulate active transport systems involved in colonic oxalate secretion.

43. The method of claim 28, wherein the Oxalobacter lysate and Oxalobacter enzyme composition locally stimulate intraluminal oxalate-degradation in intestines.

44. The method of claim 28, wherein the patient is a mammal.

45. The method of claim 28, wherein the renal disease is associated with oxalate levels in the kidneys.

46. The method of claim 28, wherein the method is used to treat a patient with hyperoxaluria.

47. The method of claim 28, wherein the lysate and enzyme composition is derived from Oxalobacter formigenes.

48. The method of claim 28, wherein treatment of the patient further comprises administering to the patient oxalate-degrading enzymes.

49. The method of claim 28, wherein the oxalate-degrading enzymes are formyl- CoA transferase and oxalyl CoA decarboxylase.