BY INHIBITING UDP-HEXOSE SIGNALING

Cross-Reference to Related Applications

This application claims the benefit of, and priority to, U.S. Provisional Application No. 62/675,256, filed May 23, 2018, the contents of which are incorporated herein by reference.

Field of the Invention

The invention relates to methods of treating and preventing renal inflammation.

Background

Over 2 million people die each year from kidney failure. Kidney failure includes both acute kidney injury (AKI) and chronic kidney disease (CKD), each of which can have any of several causes. A primary reason kidney failure is so deadly is that existing treatments are inadequate. For example, AKI has many causes, and treatment of AKI depends on identification and treatment of the underlying cause. Thus, no broadly-applicable AKI therapeutic exists. In addition, some strategies designed to prevent AKI, such as high-dose perioperative atorvastatin and remote ischemic preconditioning, have shown little proven benefit. Therapies for CKD include angiotensin converting enzyme inhibitors (ACEIs) or angiotensin II receptor antagonists (ARBs), but patients on these medications still exhibit progressive loss of kidney function. Consequently, kidney failure continues to prematurely claim the lives of millions of people worldwide each year.

Summary

The invention provides compositions and methods for preventing kidney failure by treating or preventing renal inflammation. The invention recognizes that uncontrolled inflammation is a leading cause of kidney failure and that elevated levels of UDP-hexoses (such as UDP-glucose, UDP-galactose, UDP-glucuronic acid, N-acetyl-UDP-glucosamine and/or N- acetyl-UDP-galactosamine) trigger renal inflammation via binding of a UDP-hexose to a P2Y 14 receptor associated with renal tissue. The invention further recognizes that reducing or preventing the binding of UDP-hexoses to P2Y 14 would reduce or prevent renal inflammation. Accordingly, the invention provides compositions and methods for reducing the concentration of UDP-hexoses or altering the structure of UDP-hexoses so that they cannot bind to P2Y 14. In that manner, the invention reduces or eliminates binding of UDP-hexoses to P2Y 14 and prevents development of renal inflammation. Consequently, the methods allow prevention of kidney failure in patients suffering from either acute or chronic kidney conditions.

Aspects of the invention include reducing the concentration of a UDP-hexose in a subject. Reduction of UDP-hexose concentration may be accomplished by providing a reagent that promotes degradation of the UDP-hexose or conversion of the UDP-hexose to a compound that does not promote renal inflammation. For example, the reagent may be an enzyme that degrades or modifies the UDP-hexose. Alternatively or additionally, the reagent may be a chelating agent, such as an agent that sequesters divalent cations to promote enzymatic degradation or modification of the UDP-hexose. Alternatively or additionally, the reagent may be an antibody (polyclonal or monoclonal) or other protein that binds and sequesters the UDP- hexose(s).

The methods may include providing multiple reagents that promote degradation of the UDP-hexose or conversion of the UDP-hexose to a different compound or sequestration of the UDP-hexoses. The multiple reagents may be provided simultaneously or sequentially.

The methods may include providing a reagent that reduces the concentration of a UDP- hexose in combination with another therapeutic agent that treats a disease, disorder, or condition that is associated with kidney failure, AKI, or CKD or may cause kidney failure, AKI, or CKD.

The methods may include measuring the concentration of UDP-hexose in a sample from the subject. The methods may include making multiple measurements of the concentration of UDP-hexose. The measurements may be taken before or after providing a reagent that promotes degradation of the UDP-hexose or conversion of the UDP-hexose to a different compound or sequestration of the UDP-hexoses. The methods may include taking at least one measurement before providing the reagent and taking at least one measurement after providing the reagent.

The UDP-hexose may be any UDP-hexose associated with renal inflammation. For example, the UDP-hexose may be UDP-glucose, UDP-galactose, UDP-glucuronic acid, N- acetyl-UDP-glucosamine, or N-acetyl-UDP-galactosamine.

In another aspect, methods of the invention include altering the structure or availability of a UDP-hexose in a subject. Alteration of the structure of the UDP-hexose may be accomplished by providing a reagent that changes the conformation of the UDP-hexose. For example, the reagent may be an antibody that binds to the UDP-hexose. The reagent may change the conformation of the UDP-hexose. Alternatively or additionally, the reagent may alter the availability of the UDP-hexose to serve as a reactant or to bind to other molecules. The UDP- hexose may be any UDP-hexose associated with renal inflammation, such as those described above.

Detailed Description

The invention provides methods of treating and preventing renal inflammation. Kidney failure can result from a variety of primary conditions but is nearly always a consequence of renal inflammation. For example, renal inflammation contributes to the development and progression of acute kidney injury (AKI) and is also correlated with morbidity in patients with chronic kidney disease. The invention recognizes that alleviating renal inflammation can prevent kidney failure even if the underlying cause of impaired kidney function cannot be remedied or is unknown.

Recent reports have identified the purinergic receptor P2Y 14, also called GPR105, as a key mediator of renal inflammation. The gene and protein for human P2Y 14 are described in, for example, Entrez Gene ID no. 9934, GenBank ID no. D13626, RefSeq ID no. NM_0l4879, and UniProt ID no. NM_0l487, the contents of which are incorporated herein by reference.

P2Y 14 is a G protein-coupled receptor expressed on the surface of intercalated cells (ICs) in the collecting duct system of the kidney. P2Y 14 binds uridine diphosphate glucose (UDP-glucose), an ester of pyrophosphoric acid with the nucleoside uridine, and other UDP-hexoses, such as UDP-galactose, UDP-glucuronic acid, N-acetyl-UDP-glucosamine, and N-acetyl-UDP- galactosamine. Abbracchio et ah, Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family, Trends Pharmacol Sci. 2003 Feb;24(2):52-5, DOI: l0.l0l6/S0l65-6l47(02)00038-X, the contents of which are incorporated herein by reference. UDP-glucose is released into extracellular fluids from damaged cells and in a regulated manner from intact cells. Binding of UDP-glucose to P2Y 14 triggers ICs to produce chemokines that lead to infiltration of neutrophils into the renal medulla. See Azroyan et ah, Renal Intercalated Cells Sense and Mediate Inflammation via the P2Y14 Receptor, PLoS ONE 10(3): e0121419 (2015), doi:l0.l37l/joumal.pone.0l2l4l9. Thus, high levels of UDP-glucose activate P2Y 14 to cause renal inflammation.

The invention recognizes that renal inflammation in a subject can be mitigated by decreasing the availability of UDP-hexoses to bind to P2Y 14. Thus, the invention provides methods that reduce the effective levels of UDP-hexoses, thereby attenuating P2Y 14 signaling. Certain embodiments of the invention entail degrading UDP-hexoses, converting them into other compounds that are unable to activate P2Y 14, or otherwise rendering them unavailable to bind to P2Y 14. Other embodiments involve changing the structure of UDP-hexoses to inhibit their ability to bind to P2Y 14.

Methods of reducing UDP-hexose concentration or altering UDP-hexose structure

The invention provides methods of reducing the concentration of one or more UDP- hexoses, such as UDP-glucose, UDP-galactose, UDP-glucuronic acid, N-acetyl-UDP- glucosamine, and N-acetyl-UDP-galactosamine, that stimulate activity of P2Y14 in subject. UDP-hexoses are synthesized by uridylyltransferases that reversibly transfer uridine

monophosphate (UMP) moieties from UTP to a hexose. Examples of uridylyltransferases include UTP— glucose- 1 -phosphate uridylyltransferase, which is described in, for example, Entrez Gene ID no. 7360, GenBank ID no. BC047004, and RefSeq ID no. NM_006759, and UTP— hexose- 1 -phosphate uridylyltransferase, which is described in, for example, Entrez Gene ID no. 2592, GenBank ID no. NC_000009, and RefSeq ID no. NM_00l258332. UDP-hexoses are broken down by enzymes that remove UMP moieties from the hexose. Examples of such enzymes include UDP-glucose pyrophosphatase, also called UGPPase, described in, for example, Entrez Gene ID no. 256281, GenBank ID no. BC041584, and RefSeq ID no.

NM_l77533, and uridine 5'-diphospho-glucuronosyltransferase, also called UDP- glucuronosyltransferase or UGT, described in, for example, Entrez Gene ID no. 54658, GenBank ID no. DQ364247, and RefSeq ID no. NM_000463. Other enzymes involved in UDP-hexose metabolism include UDP— glucose— hexose- 1 -phosphate uridylyltransferases, UDP— N- acetylgalactosamine diphosphorylases, UDP— N-acetylglucos amine diphosphorylases, UTP— monosaccharide- 1 -phosphate uridylyltransferase, and UTP— xylose- l-phosphate

uridylyltransferases . Therefore, the concentration of a UDP-hexose can be reduced by providing a subject with a reagent that inhibits formation of a UDP-hexose or facilitates the degradation of a UDP- hexose. For example, the reagent may be an enzyme, such as one of the aforementioned enzymes, that converts a UDP-hexose into another molecule that does not activate P2Y 14.

Uridylyltransferases may be suitable for this purpose because they catalyze both addition and removal of UMP depending on environmental conditions, and an excess of UDP-hexose can shift the reaction in favor of UMP removal.

Another suitable reagent is an agent that chelates divalent cations. Many of the aforementioned enzymes require divalent cations, such as Mg ²⁺ or Zn ²⁺ , for catalytic activity. Consequently, chelators can attenuate synthesis of UDP-hexoses by preventing cations from serving as cofactors in the enzymatic reactions. Examples of chelating agents include, without limitation, ZX1 ((2-((Bis(pyridin-2-ylmethyl)amino)methylamino)benzenesulfon ic acid; Pan, et al., Neuron 2011, 71, 1116-1126.), ZX1E, TPA (Tris[(2-pyridyl)methyl]amine), phanquinone (4,7-phenanthroline-5,6-dione), clioquinol (PN Gerolymatos SA), chloroquinol, penicillamine, trientine, N,N'-diethyldithiocarbamate (DDC), 2,3,2'-tetraamine (2,3,2'-tet), neocuproine, N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), l,lO-phenanthroline (PHE), tetraethylenepentamine (TEPA), triethylene tetraamine and tris(2-carboxyethyl)phosphine (TCEP), bathophenanthroline disulfonic acid (BPADA), ethylenediaminetetraacetic acid

(EDTA), ethylene glycol (bis) aminoethyl ether tetra acetic acid (EGTA), nitrilotriacetic acid, N,N-bis(2-hydroxyethyl)glycine (bicine); 0,0'-bis(2-aminophenyl ethylene glycol)

ethylenediamine-N,N,N',N'-tetraacetic acid (BAPTA), trans-l, 2-diamino cyclohexane- ethylenediamine-N,N,N',N'-tetraacetic acid (CyDTA), l,3-diamino-2-hydroxy-propane- ethylenediamine-N,N,N', N'-tetraacetic acid (DPTA-OH), ethylene-diamine-N,N'-dipropionic acid dihydrochloride (EDDP), ethylenediamine-N,N'-bis(methylenephosphonic acid)

hemihydrate (EDDPO), ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid)

(EDTPO), N,N'-bis(2-hydroxybenzyl)ethylene diamine-N,N'-diacetic acid (HBED), 1,6- hexamethylenediamine-N,N,N', N'-tetraacetic acid (HDTA, or HEDTA), N-(2- hydroxyethyl)iminodiacetic acid (HID A), iminodiacetic acid (IDA), l,2-diaminopropane- N,N,N', N'-tetraacetic acid (methyl-EDTA), nitriltriacetic acid (NTA), nitrilotripropionic acid (NTP), nitrilotris (methylenephosphonic acid) trisodium salt (NTPO), triethylenetetramine- N,N,N',N'',N''-hexaacetic acid (TTHA), bathocuproine, bathophenanthroline, TETA, citric acid, salicylic acid, and malic acid, and analogues and derivatives, including hydrophobic derivatives and pharmaceutically acceptable salts thereof. A combination of two or more chelating agents may also be used. Chelating agents are described in, for example, U.S. Publication No.

2014/0179741.

Another class of reagents that can reduce the effective concentration of a UDP-hexose includes agents that bind to a UDP-hexose and prevent it from activating P2Y 14. Any suitable binding agent may be used. The binding agent may be an antibody. As used herein, "antibody" encompasses complete antibody molecules, fragments of antibody molecules, and antibody- derived molecules that specifically bind an antigen. For example and without limitation, an antibody may be a complete immunoglobulin, antigen-binding fragment (Fab), Fab2, variable domain (Fv), single chain variable fragment (scFv), third-generation (3G) antibody. The antibodies may be natural monoclonal antibodies or synthetic antibodies, such as recombinant antibodies, non-immunoglobulin derived synthetic antibodies, or affimer proteins. Methods of making monoclonal antibodies are known in the art and described in, for example, Antibodies: A Laboratory Manual, Second edition, edited by Greenfield, Cold Spring Harbor Laboratory Press (2014) ISBN 978-1-936113-81-1. Methods of making synthetic antibodies are described in, for example, US 2014/0221253; US 2016/0237142; and Miersch and Sidhu, Synthetic antibodies: concepts, potential and practical considerations, Methods. 2012 Aug;57(4):486-98. doi:

10. l0l6/j.ymeth.2012.06.012, the contents of each of which are incorporated herein by reference.

Another type of UDP-hexose binding agent is a version of an enzyme that uses the UDP- hexose as a substrate but has been modified to eliminate its catalytic activity. Any enzyme that binds a UDP-hexose may be used. For example and without limitation, the enzyme may be a UDP— glucose— hexose-l -phosphate uridylyltransferase, UDP— N-acetylgalactosamine diphosphorylase, UDP— N-acetylglucosamine diphosphorylase, UTP— glucose- 1 -phosphate uridylyltransferase, UTP— hexose-l -phosphate uridylyltransferase, UTP— monosaccharide- 1- phosphate uridylyltransferase, or UTP— xylose- 1 -phosphate uridylyltransferase.

The invention also provides methods of altering the structure of one or more UDP- hexoses, such as UDP-glucose, UDP-galactose, UDP-glucuronic acid, N-acetyl-UDP- glucosamine, and N-acetyl-UDP-galactosamine, that stimulate activity of P2Y14 in subject. The structure of the UDP-hexose may be altered by providing a reagent that binds to the UDP-hexose to alter its conformation. Any suitable reagent may be used. For example and without limitation, the reagent may be an antibody, an enzyme (including a modified version of an enzyme), a chelator, an ion, or a small molecule.

Kidney failure, AKI, and CKD

Kidney failure, also called end-stage renal disease, generally refers to a state or condition when the kidneys no longer function well enough for the subject to survive without dialysis or a kidney transplant. Kidney failure may be classified based on function of the failing kidneys in relation to normal kidney function. Commonly, a subject having kidney function below 15% of normal is considered to have kidney failure.

Kidney failure may result from, or be a stage of, acute kidney injury (AKI) or chronic kidney disease (CKD). AKI is an abrupt loss of kidney function that develops within 7 days. CKD is the gradual loss of kidney function over a period of months or years.

AKI may be assessed by any suitable standard. Several standards for acute kidney injury are known in the art, such as the criteria provided by the Acute Kidney Injury Network (AKIN); Kidney Disease Improving Global Outcomes (KDIGO); and Risk, Injury, Failure, Loss, and End-stage Kidney (RIFLE). AKI may be categorized or staged according to the AKI, KDIGO, or RIFLE criteria. For example, a subject may be deemed to have stage 1, stage 2, or stage 3 AKI, or a subject may be deemed to have risk, injury, failure, or loss. The standard may apply to an adult, pediatric, newborn, neonatal, infant, child, adolescent, pre-teen, teenage, or elderly subject.

Standards typically include measurements of serum creatinine (SCr) concentrations, urine output, or glomerular filtration rate (GFR). Standards may include multiple parameters, e.g., combinations of the aforementioned standards. A subject may be deemed to have AKI, or a stage or category thereof, when she has abnormally high SCr concentration, abnormally low urine output, abnormally low GFR, or any combination thereof. Standards may be absolute, e.g., they may require a value above or below a defined threshold value. Alternatively, standards may be relative, e.g., they may require an increase or decrease relative to a baseline value. Standards for different parameters, e.g., abnormally high SCr concentration abnormally low urine output, or abnormally low GFR, may independently be absolute or relative. Standards for acute kidney injury may include a temporal component. For example, a subject may be deemed to have AKI when an elevated SCr concentration is measured at some interval following a preceding event. The preceding event may be cardiac surgery, cardiac arrest, admission to a hospital, clinic, medical facility, or any unit thereof. The interval may be 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours. A subject may be deemed to have AKI when urine output is measured across some interval, such as 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours.

For example and without limitation, a standard for reduced urine output associated with AKI may be less than 0.5 mL/kg/h for 6-12 hours, less than 0.5 mL/kg/h for at least 12 hours, or less than 0.3 mL/kg/h for 24 hours, or anuria for at least 12 hours.

For example and without limitation, a standard for elevated SCr concentration associated with AKI may be a SCr concentration of at least 0.3 mg/dl, a SCr concentration of at least 1 mg/dl, a SCr concentration of at least 4 mg/dl, a SCr concentration of at least 26.5 pmol/l, or a SCr concentration of at least 353.6 pmol/l. For example and without limitation, a standard for elevated SCr concentration associated with AKI may be an increase of 50% over baseline, an increase of 100% over baseline, or an increase of 200% over baseline.

For example and without limitation, a standard for GFR associated with AKI may be a GFR of less than 35 ml/min per 1.73 mm . For example and without limitation, a standard for GFR associated with AKI may be a decrease of at least at least 25% relative to a baseline, a decrease of at least at least 50% relative to a baseline, or a decrease of at least at least 75% relative to a baseline.

AKI may be secondary to, or associated with, another disease, disorder, or condition. For example, AKI may result from, or be associated with, low blood pressure, surgery, a cardiac event (such as a heart attack), a cerebrovascular event (such as stroke or subarachnoid hemorrhage), a trauma, an infection, blockage of the urinary tract, muscle breakdown, or hemolytic uremic syndrome.

CKD may be staged based on glomerular filtration rate (GFR), expressed in units of ml/min/l.73 m ² . Individuals with Stage 1 CKD have a GFR of > 90 and display slightly diminished function, kidney damage, and persistent albuminuria. Kidney damage may be defined as pathological abnormalities or markers of damage, including abnormalities in blood or urine tests or imaging studies. Individuals with Stage 2 CKD have a GFR of 60-89 and display kidney damage. Individuals with Stage 3 CKD have a GFR of 30-59. Individuals with Stage 4 CKD have a GFR of 15-29. Individuals with Stage 5 CKD, also called end-stage kidney disease (ESKD) have a GFR of < 15 and usually require renal replacement therapy, i.e., dialysis or kidney transplantation. Non-dialysis dependent chronic kidney disease (NDD-CKD)

encompasses individuals with established CKD who do not yet require renal replacement therapy.

CKD may be secondary to, or associated with, another disease, disorder, or condition.

For example, AKI may result from, or be associated with diabetes, high blood pressure, nephrotic syndrome, glomerulonephritis, or polycystic kidney disease.

Methods of providing a reagent

The invention provides methods of providing a reagent to a subject. Providing the reagent to the subject may include administering it to the subject. The reagent may be administered by any suitable means. For example and without limitation, the reagent may be administered orally, intravenously, enterally, parenterally, dermally, buccally, topically, transdermally, by injection, intravenously, subcutaneously, nasally, pulmonarily, or with or on an implantable medical device (e.g., stent or drug-eluting stent or balloon equivalents).

The reagent may be provided at any suitable dosage. For example and without limitation, the reagent may be provided at from 0.001 mg/kg body weight to 5 g/kg body weight. In some embodiments, the dosage range is from 0.001 mg/kg body weight to 1 g/kg body weight, from 0.001 mg/kg body weight to 0.5 g/kg body weight, from 0.001 mg/kg body weight to 0.1 g/kg body weight, from 0.001 mg/kg body weight to 50 mg/kg body weight, from 0.001 mg/kg body weight to 25 mg/kg body weight, from 0.001 mg/kg body weight to 10 mg/kg body weight, from 0.001 mg/kg body weight to 5 mg/kg body weight, from 0.001 mg/kg body weight to 1 mg/kg body weight, from 0.001 mg/kg body weight to 0.1 mg/kg body weight, or from 0.001 mg/kg body weight to 0.005 mg/kg body weight. Alternatively, in some embodiments the dosage range is from 0.1 g/kg body weight to 5 g/kg body weight, from 0.5 g/kg body weight to 5 g/kg body weight, from 1 g/kg body weight to 5 g/kg body weight, from 1.5 g/kg body weight to 5 g/kg body weight, from 2 g/kg body weight to 5 g/kg body weight, from 2.5 g/kg body weight to 5 g/kg body weight, from 3 g/kg body weight to 5 g/kg body weight, from 3.5 g/kg body weight to 5 g/kg body weight, from 4 g/kg body weight to 5 g/kg body weight, or from 4.5 g/kg body weight to 5 g/kg body weight. These doses may be administered at a single time, at multiple times, or continuously, for example, by continuous intravenous infusion. Multiple doses may be administered at any interval. For example, the interval between doses may be about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 1 day, about 2 days, about 3 days, or more. Effective doses may be estimated from dose-response relationships derived from in vitro or animal model test bioassays or systems or from clinical trials of the P2Y 14 antagonist. The dosage should not be so large as to cause unacceptable adverse side effects.

The reagent may be provided in combination with another therapeutic agent that treats or prevents kidney failure, CKD, AKI, or an underlying cause of any of the aforementioned conditions. For example and without limitation, the reagent may be provided in combination with one or more of an angiotensin converting enzyme inhibitor (ACEI), angiotensin II receptor antagonist (ARB), cyclophosphamide, diuretic, such as furosemide, inotrope, such as dobutamine, intravenous fluid, phosphate binder, phosphodiesterase-5 inhibitor, steroid, vasopressors, such as norepinephrine, or zinc. The reagent and one or more additional therapeutic agents may be provided simultaneously as part of a single formulation or in separate formulations. The reagent and one or more additional therapeutic agents may be provided at different times and/or according to independent dosing schedules.

Measuring UDP-hexose levels

The methods of the invention may include measuring levels of a UDP-hexose, such as UDP-glucose, UDP-galactose, UDP-glucuronic acid, N-acetyl-UDP-glucosamine, and N-acetyl- UDP-galactosamine, in subject. The level of a UDP-hexose may be measured by any suitable method. Preferably, UDP-glucose is measured by coupling a reaction converting UDP-glucose to a byproduct with the stoichiometric production of NADH or UDP as described in WO 2017/165665, the contents of which are incorporated herein in their entirety. UDP-glucose levels may also be measured using the protocols described in Barrett et ah, Molec. Pharmacol., 2013, 84, 41-49, the contents of which are incorporated herein by reference in their entirety. Alternatively, UDP-glucose levels may be measured by using an anti-UDP-glucose antibody.

In certain embodiments that entail measurement of NADH as the readout molecule, the assay may include pre-processing steps to remove proteins that interact with NADH production and/or endogenous NADH from the sample. High levels (e.g., > 2 mM) of endogenous NADH from the sample can inhibit the assay. Alternatively or additionally, a control reaction lacking exogenous enzyme may be performed to measure the amount of pre-existing NADH in the sample, and this value can be subtracted from the value obtained from a reaction that receives exogenous enzyme. Next, the liquid sample is buffered to pH 8-9, for example, pH 8.0. The enzyme UDP-glucose dehydrogenase is added to the reaction along with the co-factor NAD ⁺ . During the reaction UDP-glucose is converted to UDP-glucuronic acid, and a stoichiometric amount of NAD ⁺ is converted to NADH. NADH is then measured, and its concentration is used to deduce the starting UDP-glucose concentration. The amount of substrate and/or the reaction rate may be optimized so that the reaction occurs substantially in a substantially linear portion of the Michaelis-Menten graph.

In some embodiments, excess NAD ⁺ is added to the reaction, along with enzyme in excess, such that UDP-glucose is limiting. For example, NAD ⁺ can be added to a concentration of 2 mM per well, and 0.04 units of enzyme added per well to achieve an excess of both. One unit of enzyme is the amount of UDP-glucose dehydrogenase required to oxidize 1.0 pmole of UDP-glucose to UDP-glucuronic acid per minute at pH 8.7 at 25°C.

Alternatively, the complete reaction curve can be determined for each sample and the data fit to a non-linear rate equation (e.g.,“progress-curve analysis”). This is particularly useful when the slope of the linear region of the Michaelis-Menten kinetics curve for a desired enzyme is very steep (e.g., when the initial rate is too fast to measure accurately) or when an excess of substrate (e.g., NAD ⁺ ) is used in the reaction mix

The methods may include lateral flow assays adapted for use in the detection of NADH or UDP. Such lateral flow assays permit the flow of a liquid sample, applied to the sample application zone, to deliver the sample/reactants to a test region (e.g., a reaction zone) of the lateral strip or device, and then the sample with a generated byproduct is delivered to a detection zone, which provides a readout (e.g., visual, optical, fluorescent, etc.). As one example, an assay may use reduction of nitro blue tetrazolium (NBT) by NADH to generate a colored product at a test region. As samples with generated NADH flow over a region with NBT (no color), the NBT is reduced to the blue form, which is visible on a strip

In some embodiments in which NBT is used to generate a detectable product, a reductase may be immobilized on the dipstick or test strip. The reductase may be a diaphorase, and it may be immobilized via adsorption or via immunocapture. As the NADH-containing solution flows through the region with the reductase enzyme, the NADH is oxidized and would reduce the NBT to the colored precipitate NBTH.

In some embodiments, the level of NADH or UDP in a sample is detected by a lateral flow assay test (LFA), or strip test. LFAs detect the presence or absence of an analyte, e.g.

NADH or UDP, in a liquid sample. With a lateral flow method, a spatial separation is defined in the strips between the sample application zone and detection region. Most conventional lateral flow strips are designed for test samples that are readily available in large quantities (e.g., urine). Lateral flow immunoassays are described below, but lateral flow assays may also be adapted for the measurement of an analyte without the use of antibody. Both lateral flow immunoassays (e.g., using a UDP-glucose antibody) and lateral flow analyte assays (e.g., detection of NADH to measure UDP-glucose levels) are contemplated for use herein.

In LFAs the test sample flows along a solid substrate via capillary action. After the sample is applied to the lateral flow strip, it encounters a test region where an enzymatic reaction coupled to NADH or UDP production occurs and continues to a region comprising a detection reagent that permits detection of NADH or UDP. The liquid may go through one or more different regions on the lateral flow strip following the test region and prior to the detection region.

LFAs are adapted to operate along a single axis to suit the test strip format or a dipstick format. Typically, LFAs proceed from sample application to readout without additional steps by the user, so sample application generally leads to an assay result with the further user input.

Other lateral flow configurations may include one or more steps by the user after sample application, e.g., insertion into a detector device (e.g., a luminometer, fluorescence detector, etc.) or addition of another reagent. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens or analytes from fluid samples such as urine, blood, water samples etc. Strip tests are also known as "dipstick tests," the name bearing from the literal action of "dipping" the test strip into a fluid sample to be tested. LFA strip tests are easy to use, require minimum training and can easily be included as components of point- of-care test (POCT) diagnostics to be used on site in the field.

A typical test strip may comprise one or more of following components: (1) sample application zone comprising e.g., an absorbent pad (i.e., the matrix or material) onto which the test sample is applied; (2) test region comprising immobilized enzyme; (3) a test results area comprising a detection reagent or reaction membrane - such as a hydrophobic nitrocellulose or cellulose acetate membrane onto which, for example, a detection reagent is immobilized in a line across the membrane as a capture zone or test line (a control zone may also be present, containing NADH or another reducing agent, for example, that reduces NBT to generate a blue color) or an antibody reagent; and (4) optional wick or waste reservoir - a further absorbent pad designed to draw the sample across the detection reagent zone or reaction membrane by capillary action and collect it. In addition, lateral flow strips as described herein may further comprise one or more of the following: a region comprising a strong base or a region comprising immobilized NAD ⁺ nucleosidase to degrade unreacted NAD ⁺ .

The components of the strip may be fixed to an inert backing material and may be presented in a simple dipstick format or within a plastic casing with a sample port and reaction window showing the test readout/capture and control zones. The test may incorporate a second, coated line which contains an antibody or other reagent that picks up free readout substrate (e.g., free latex or gold particles) in order to confirm the test has operated correctly.

The use of "dip sticks" or LFA test strips and other solid supports has been described in the art in the context of an immunoassay for a number of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; and 6,565,808, and U.S. patent applications Ser. Nos. 10/278,676; 09/579,673; and 10/717,082, which are incorporated herein by reference in their entirety, are non-limiting examples of such lateral flow test devices.

Examples of patents that describe the use of "dip stick" technology to detect soluble antigens via immunochemical assays include, but are not limited to, U.S. Patent Nos. 4,444,880; 4,305,924; and 4,135,884; which are incorporated by reference herein in their entireties. The apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a "dip stick" which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the "dip stick," prior to detection of the component- antigen complex upon the stick. Given the reaction description and considerations described herein, it is within the skill of one in the art to modify the teachings regarding "dip stick" technology for the detection of NADH or UDP using e.g., dye, luciferin or fluorescent reagents as described herein.

In some embodiments, the reaction to generate a stoichiometric amount of NADH from the reaction of UDP-glucose with UDP-glucose dehydrogenase is incubated for a matter of minutes, e.g., 5 or 10 minutes, in the liquid assay format in order to generate sufficient amounts of NADH for detection. This extended time is not as readily achieved in the dipstick or lateral flow format. However, options to overcome this include performing the first enzymatic reaction in an assay well for a prescribed period of time before inserting a dipstick or applying sample to a test strip. Alternatively, if all reactions took place on the dipstick or test strip, a shorter incubation should not present a problem because most of the enzyme reaction actually takes place within the first minute, although the reaction continues to remain linear after a 5 -minute incubation, after the initial linear velocity for low (physiological) concentrations of UDP-glucose (up to 100 mM).

A urine dipstick is a colorimetric chemical assay comprising a reagent stick-pad. The dipstick is typically immersed in a fresh urine specimen and then withdrawn. Alternatively, the urine sample may be applied directly to the sample application zone by the subject (e.g., analogous to a pregnancy test). After predetermined times the colors of the reagent pad are compared to standardized reference charts. The urine dipstick offers an inexpensive and fast method to perform screening urinalyses, which helps in identifying the presence of various diseases or health problems. A urine dipstick provides a simple and clear diagnostic guideline and may be used in the methods and kits as described herein. Accordingly, one aspect of the present technology relates to a method for detecting NADH or UDP using a device, such as a dipstick, as described herein. When the sample is not clear, a centrifugation or filtration step to render a clear sample may be applied so as to avoid pigment or other entities from fouling the optical readout.

In some cases, the lateral flow strip may also comprise a control that gives a signal to the user that the assay is performing properly. For instance, the control zone may contain an immobilized receptive material that is generally capable of forming a chemical and/or physical bond with probes or with the receptive material immobilized on the probes. Some examples of such receptive materials include, but are not limited to, antigens, haptens, antibodies, protein A or G, avidin, streptavidin, secondary antibodies, and complexes thereof. In addition, it may also be desired to utilize various non-biological materials for the control zone receptive material. For instance, in some embodiments, the control zone receptive material may also include a polyelectrolyte that may bind to uncaptured probes. Because the receptive material at the control zone is only specific for probes, a signal forms regardless of whether the analyte is present. The control zone may be positioned at any location along the test strip, but is preferably positioned downstream from the detection zone.

In some embodiments, detection involves reduction of nitro blue tetrazolium by NADH present and/or generated during the assay. In such embodiments, the control line may include a line of NBT spatially downstream of the test line and immediately downstream of a line or zone of dried reducing agent. Flow of sample past the test line will liberate the reducing agent and carry it to the control line of NBT, which will be reduced to generate a control line indicating the sample reactants have successfully reacted at that point.

Qualitative, semi-quantitative, and quantitative results may be obtained with the lateral flow assays described herein. For example, when it is desired to semi-quantitatively or quantitatively detect an analyte, the intensity of any signals produced at the region comprising a detection reagent may be measured with e.g., an optical reader. The actual configuration and structure of the optical reader may generally vary as is readily understood by those skilled in the art. For example, optical detection techniques that may be utilized include, but are not limited to, luminescence (e.g., fluorescence, phosphorescence, etc.), absorbance (e.g., fluorescent or non- fluorescent), diffraction, etc. Further optical methods include but are not limited to, measurement of light scattering or simple reflectance, e.g., using a luminometer or photomultiplier tube;

radioactivity, e.g., using a Geiger counter; electrical conductivity or dielectric capacitance; and release of electroactive agents, such as indium, bismuth, gallium or tellurium ions.

Once the amount of detection agent has been quantified, the amount may then be mapped onto another measurement scale. For example, while the result of the assay may be measured as a density of reflectance (Dr), the result reported may be more meaningful in other units, such as RI (intensity relative to that of a control zone or background level). Results may also be expressed as the number of copies of analyte present in the measurement volume.

The methods may include lateral flow immunoassays (LFIAs), in which antibodies that bind a target analyte are used in a competitive or sandwich immunoassay adapted to the lateral flow format. Conventional sandwich LFIAs are similar to sandwich ELIS As. The sample first encounters and mobilizes colored particles which are labeled with antibodies raised to the target antigen. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen. The test line will show as a colored band in positive samples, resulting from the accumulation or capture of antibody-bearing colored particles. In some embodiments, the lateral flow immunoassay may be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof. Conventional competitive LFIAs are similar to competitive ELISA. The sample first encounters colored particles which are labeled with the target antigen or an analogue. The test line contains antibodies to the target/its analogue. Unlabeled antigen in the sample will block the binding sites on the antibodies preventing capture of the colored particles at the test line. The test line will show as a colored band in negative samples. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test.

Any substance generally capable of producing a signal that is detectable visually or by an instrumental device may be used as a detection reagent. Suitable detectable substances may include, for instance, luminescent compounds (e.g., fluorescent, phosphorescent, etc.);

radioactive compounds; visual compounds (e.g., colored dye or metallic substance, such as gold); liposomes or other vesicles containing signal-producing substances; enzymes and/or substrates, and so forth. Other suitable detectable substances are described in U.S. Pat. Nos. 5,670,381 and 5,252,459, which are incorporated herein in their entirety by reference. If the detectable substance is colored, the ideal electromagnetic radiation is light of a complementary wavelength. For instance, blue detection probes strongly absorb red light.

In some embodiments, the detectable substance may be a luminescent compound that produces an optically detectable signal. For example, suitable fluorescent molecules may include, but are not limited to, fluorescein, europium chelates, phycobiliprotein, rhodamine, and their derivatives and analogs. Other suitable fluorescent compounds are semiconductor nanocrystals commonly referred to as "quantum dots."

In another embodiment, the detection agent is a particle. Examples of particles useful in the methods, assays and kits described herein include, but are not limited to, colloidal gold particles; colloidal sulfur particles; colloidal selenium particles; colloidal barium sulfate particles; colloidal iron sulfate particles; metal iodate particles; silver halide particles; silica particles; colloidal metal (hydrous) oxide particles; colloidal metal sulfide particles; colloidal lead selenide particles; colloidal cadmium selenide particles; colloidal metal phosphate particles; colloidal metal ferrite particles; any of the above-mentioned colloidal particles coated with organic or inorganic layers; protein or peptide molecules; liposomes; or organic polymer latex particles, such as polystyrene latex beads. Further, suitable phosphorescent compounds include metal complexes of one or more metals, such as ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, iron, chromium, tungsten, zinc, and so forth. Especially preferred are ruthenium, rhenium, osmium, platinum, and palladium. The metal complex may contain one or more ligands that facilitate the solubility of the complex in an aqueous or non- aqueous environment. For example, some suitable examples of ligands include, but are not limited to, pyridine; pyrazine; isonicotinamide; imidazole; bipyridine; terpyridine;

phenanthroline; dipyridophenazine; porphyrin, porphine, and derivatives thereof. Such ligands may be, for instance, substituted with alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino, hydroxycarbonyl, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing groups, phosphorus containing groups, and the carboxylate ester of N-hydroxy-succinimide.

Porphyrins and porphine metal complexes possess pyrrole groups coupled together with methylene bridges to form cyclic structures with metal chelating inner cavities. Many of these molecules exhibit strong phosphorescence properties at room temperature in suitable solvents (e.g., water) and an oxygen-free environment. Some suitable porphyrin complexes that are capable of exhibiting phosphorescent properties include, but are not limited to, platinum (II) coproporphyrin- 1 and II, palladium (II) coproporphyrin, ruthenium coproporphyrin, zinc(II)- coproporphyrin-I, derivatives thereof, and so forth. Similarly, some suitable porphine complexes that are capable of exhibiting phosphorescent properties include, but not limited to, platinum(II) tetra-meso-fluorophenylporphine and palladium(II) tetra-meso-fluorophenylporphine. Still other suitable porphyrin and/or porphine complexes are described in U.S. Pat. Nos. 4,614,723;

5,464,741; 5,518,883; 5,922,537; 6,004,530; and 6,582,930, which are incorporated herein in their entirety by reference.

Bipyridine metal complexes may also be utilized as phosphorescent compounds. Some examples of suitable bipyridine complexes include, but are not limited to, bis [(4,4'- carbomethoxy)-2,2'-bipyridine]2-[3-(4-methyl-2,2'-bipyridine -4-yl)propyl]-l,3-dioxolane ruthenium (II); bis(2,2'bipyridine)[4-(butan-l-al)-4'-methyl-2,2'-bi-pyridin e]ruthenium (II); bis(2,2'-bipyridine)[4-(4'-methyl-2,2'-bipyridine-4'-yl)-but yric acid] ruthenium (II);

tris(2,2'bipyridine)ruthenium (II); (2,2'-bipyridine)[bis-bis(l,2-diphenylphosphino)ethylene]2-[ 3- (4-methyl-2,2'-bipyridine-4'-yl)propyl]-l,3-dioxolane osmium (II); bis(2,2'-bipyridine)[4-(4'- methyl-2, 2'-bipyridine)-butylamine]ruthenium (II) ; bis(2,2'-bipyridine)[l-bromo-4(4'-methyl- 2,2'-bipyridine-4-yl)butane]ruthenium (II); bis(2,2'-bipyridine)maleimidohexanoic acid, 4- methyl-2,2'-bipyridine-4'-butylamide ruthenium (II), and so forth.

An immunoassay measures the concentration of a substance in a sample, typically a fluid sample, using the interaction of an antibody or antibodies to its antigen. The assay takes advantage of the highly specific binding of an antibody with its antigen. In some embodiments, specific binding of a UDP-hexose molecule with an anti-UDP-hexose antibody forms a UDP- hexose-antibody complex. The complex may then be detected by a variety of methods known in the art. An immunoassay also often involves the use of a detection antibody. Antibodies contemplated for use with the methods and assays described herein include an anti-UDP-glucose antibody, an anti-UDP-galactose antibody, and anti-UDP-glucuronic acid antibody, an anti-N- acetyl-UDP-glucosamine antibody, and an anti-N-acetyl-UDP-galactosamine antibody. Such antibodies may be designed and generated using methods known in the art and/or described herein.

In one embodiment, the antibody is detectably labeled or capable of generating a detectable signal. In one embodiment, the antibody is fluorescently labeled.

In some embodiments, levels of a desired biomarker or analyte (e.g., UDP-glucose or another UDP-hexose) are measured by ELISA, also called enzyme immunoassay or EIA. ELISA is a biochemical technique that detects the presence of an antibody or an antigen in a sample.

In one embodiment, an ELISA involving at least one antibody with specificity for the particular desired antigen may be performed. A known amount of sample and/or antigen is immobilized on a solid support (e.g., a polystyrene micro titer plate). Immobilization may be either non-specific (e.g., by adsorption to the surface) or specific (e.g., where another antibody immobilized on the surface is used to capture antigen or a primary antibody). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody may be covalently linked to an enzyme, or may itself be detected by a secondary antibody, which is linked to an enzyme through bio-conjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELIS As utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.

In one embodiment, a sandwich ELISA is used, where two antibodies specific for the target may be used. There are other different forms of ELISA, which are well known to those skilled in the art. Standard techniques known in the art for ELISA are described in "Methods in Immunodiagnosis", 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; and

Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem. 22: 895-904.

Antibodies or portions thereof may be used in immunoassays. For example, the immunoassay may use a complete immunoglobulin, antigen-binding fragment (Fab), Fab2, variable domain (Fv), single chain variable fragment (scFv), third-generation (3G) antibody.

The antibodies may be natural monoclonal antibodies or synthetic antibodies, such as

recombinant antibodies, non-immunoglobulin derived synthetic antibodies, or affimer proteins. Methods of making monoclonal antibodies are known in the art and described in, for example, Antibodies: A Laboratory Manual, Second edition, edited by Greenfield, Cold Spring Harbor Laboratory Press (2014) ISBN 978-1-936113-81-1. Methods of making synthetic antibodies are described in, for example, US 2014/0221253; US 2016/0237142; and Miersch and Sidhu, Synthetic antibodies: concepts, potential and practical considerations, Methods. 2012

Aug;57(4):486-98. doi: 10. l0l6/j.ymeth.2012.06.012, the contents of each of which are incorporated herein by reference.

In some embodiments, UDP-glucose, UDP-galactose, UDP-glucuronic acid, N-acetyl- UDP-glucosamine, N-acetyl-UDP-galactosamine or another molecule that serves as an indicator of UDP-glucose is detected by mass spectrometry, optionally in combination with liquid chromatography. Molecules may be ionized for mass spectrometry by any method known in the art, such as ambient ionization, chemical ionization (Cl), desorption electro spray ionization (DESI), electron impact (El), electrospray ionization (ESI), fast-atom bombardment (FAB), field ionization, laser ionization (LIMS), matrix-assisted laser desorption ionization (MALDI), paper spray ionization, plasma and glow discharge, plasma-desorption ionization (PD), resonance ionization (RIMS), secondary ionization (SIMS), spark source, or thermal ionization (TIMS). Methods of mass spectrometry are known in the art and described in, for example, U.S. Patent No. 8,895,918; U.S. Patent No. 9,546,979; U.S. Patent No. 9,761,426; Hoffman and Stroobant, Mass Spectrometry: Principles and Applications (2nd ed.). John Wiley and Sons (2001), ISBN 0- 471-48566-7; Dass, Principles and practice of biological mass spectrometry, New York: John Wiley (2001) ISBN 0-471-33053-1; and Lee, ed., Mass Spectrometry Handbook, John Wiley and Sons, (2012) ISBN: 978-0-470-53673-5, the contents of each of which are incorporated herein by reference.

In certain embodiments, a sample can be directly ionized without the need for use of a separation system. In other embodiments, mass spectrometry is performed in conjunction with a method for resolving and identifying ionic species. Suitable methods include chromatography, capillary electrophoresis-mass spectrometry, and ion mobility. Chromatographic methods include gas chromatography, liquid chromatography (LC), high-pressure liquid chromatography (HPLC), and reversed-phase liquid chromatography (RPLC). In a preferred embodiment, liquid chromatography-mass spectrometry (LC-MS) is used. Methods of coupling chromatography and mass spectrometry are known in the art and described in, for example, Holcapek and Brydwell, eds. Handbook of Advanced Chromatography /Mass Spectrometry Techniques, Academic Press and AOCS Press (2017), ISBN 9780128117323; Pitt, Principles and Applications of Liquid Chromatography-Mass Spectrometry in Clinical Biochemistry, The Clinical Biochemist

Reviews. 30(1): 19-34 (2017) ISSN 0159-8090; Niessen, Liquid Chromatography-Mass

Spectrometry, Third Edition. Boca Raton: CRC Taylor & Francis pp. 50-90. (2006) ISBN 9780824740825; Ohnesorge et ah, Quantitation in capillary electrophoresis-mass spectrometry, Electrophoresis. 26 (21): 3973-87 (2005) doi:l0.l002/elps.200500398; Kolch et ah, Capillary electrophoresis-mass spectrometry as a powerful tool in clinical diagnosis and biomarker discovery, Mass Spectrom Rev. 24 (6): 959-77. (2005) doi:l0.l002/mas.2005l; Kanu et ah, Ion mobility-mass spectrometry, Journal of Mass Spectrometry, 43 (1): 1-22 (2008)

doi:l0.l002/jms.l383, the contents of which are incorporated herein by reference.

The assays described herein may be adapted to be performed on an automated device platform that is programmed to automatically add, transfer and optionally, mix liquid samples or reaction mixtures, for example, in wells of a multiwell plate. The wells may include reagents as necessary, either added in liquid/solution form or, for example, dried or immobilized on a surface within the wells. Automated platforms that include liquid handling modules as well as detection (e.g., fluorescence, luminescence, absorbance, reflectance, etc.) modules are known to those of skill in the art. As but one non-limiting example, one might use, e.g., a Beckman Coulter AU5800 device. When adapted to an automated design, multiwell plates may include, in addition to test wells for assaying an unknown test sample, control wells including, e.g., blanks lacking enzyme or other reagents, to permit, among other things, the determination of background levels of, e.g., intermediate or surrogate indicator NADH. Other controls may include, e.g., positive control wells including a known amount of UDP-glucose; a set of separate positive control wells may include varying known amounts of UDP-glucose to establish a standard curve, e.g., over one or a plurality of orders of magnitude, that is read by the device and used to calculate amounts of UDP-glucose in the unknown test samples.

Samples

Measurement of levels of a UDP-hexose, such as UDP-glucose, UDP-galactose, UDP- glucuronic acid, N-acetyl-UDP-glucosamine, and N-acetyl-UDP-galactosamine, may include obtaining a sample from a subject. A sample may be obtained from any organ or tissue in the individual to be tested, provided that the sample is obtained in a liquid form or can be pre-treated to take a liquid form. For example and without limitation, the sample may be a blood sample, a urine sample, a serum sample, a semen sample, a sputum sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a plasma sample, a pus sample, an amniotic fluid sample, a bodily fluid sample, a stool sample, a biopsy sample, a needle aspiration biopsy sample, a swab sample, a mouthwash sample, a cancer sample, a tumor sample, a tissue sample, a cell sample, a synovial fluid sample, a phlegm sample, a saliva sample, a sweat sample, or a combination of such samples. The sample may also be a solid or semi-solid sample, such as a tissue sample, feces sample, or stool sample, that has been treated to take a liquid form by, for example,

homogenization, sonication, pipette trituration, cell lysis etc. For the methods described herein, it is preferred that a sample is from urine, serum, whole blood, or sputum.

In some embodiments, a sample is treated to remove cells or other biological

particulates. Methods for removing cells from a blood or other sample are well known in the art and may include e.g., centrifugation, sedimentation, ultrafiltration, immune selection, etc.

The subject may be a human. The subject may be a pediatric, a newborn, a neonate, an infant, a child, an adolescent, a pre-teen, a teenager, an adult, or an elderly patient. The subject may have kidney failure or be at risk of developing kidney failure. The subject may AKI or CKD, including any stage of AKI or CKD. The subject may have a disease, disorder, or condition that is associated with AKI or CKD. For example and without limitation, the subject may have diabetes, high blood pressure, low blood pressure, nephrotic syndrome, glomerulonephritis, or polycystic kidney disease, blockage of the urinary tract, muscle breakdown, or hemolytic uremic syndrome. The patient may have had a cardiac procedure, such as a mitral valve replacement, aneurysm repair, angioplasty, aorta transcatheter repair, biopsy, cardiomyoplasty, carotid endarterectomy, catheter repair, catheterization, chemical

cardioversion, congenital aortic stenosis surgery, congenital pulmonary stenosis balloon valvuloplasty, congenital pulmonary stenosis surgery, coronary artery bypass graft, electrical cardioversion, heart transplant, heart valve repair, heart valve replacement, implantation of a defibrillator, implantation of a stent, implantation of a stent, implantation of a total artificial heart, implantation of a ventricular assist device, implantation of pacemaker, pericardiectomy, pericardiocentesis, removal of implanted device, septal myectomy, surgery for pulmonary atresia, therapeutic hypothermia, or transmyocardial laser revascularization. The subject may have had a cardiac event, such as angina, myocardial infarction, thromboembolic or hemorrhagic stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, subarachnoid hemorrhage, peripheral artery disease, thromboembolic disease, traumatic brain injury, or venous thrombosis. The subject may be in dialysis, critical care, intensive care, neonatal intensive care, pediatric intensive care, coronary care, cardiothoracic care, surgical intensive care, medical intensive care, long-term intensive care, an operating room, an ambulance, a field hospital, or an out-of-hospital field setting.

Reference levels

After measuring the level of UDP-hexose in a subject, the level may be compared to a reference level. The reference level may be defined based on clinical trials that determine the concentration of UDP-glucose, UDP-galactose, UDP-glucuronic acid, N-acetyl-UDP- glucosamine, or N-acetyl-DP-galactosamine that optimally defines a cut-off point above which the likelihood of occurrence of kidney failure, AKI, or CKD is high and below which the likelihood of occurrence of kidney failure, AKI, or CKD is low.

The reference level of UDP-hexose may be defined by a statistic describing the distribution of levels in normal healthy subjects. For example, the reference level may be an average level of UDP-hexose in a sample from a normal healthy subject or a population of normal healthy subjects. The reference level of UDP-hexose may be an average level of UDP- hexose in a sample from a subject who has not had kidney failure, AKI, or CKD. The reference level of UDP-hexose may be an average level of UDP-hexose in a sample from one or more subjects who have the same underlying disease, disorder, or condition, such as diabetes, high blood pressure, low blood pressure, nephrotic syndrome, glomerulonephritis, or polycystic kidney disease, blockage of the urinary tract, muscle breakdown, or hemolytic uremic syndrome, but did not develop kidney failure, AKI, or CKD.

The reference level may be above the highest observed level of UDP-hexose in a sample from a normal healthy subject or a population of normal healthy subjects. Any level above the reference level may be deemed to be significantly different from the average level of UDP- hexose in a sample from a normal healthy subject or a population of normal healthy subjects.

The reference level may be greater than 95% of the levels observed in samples from a normal healthy subject or a population of normal healthy subjects, or it may be above the lower limit of the highest decile, quartile, or tertile of the levels observed in samples from a normal healthy subject or a population of normal healthy subjects.

The reference level may be at least one standard deviation, at least two standard deviations, or at least three standard deviations above the average level of UDP-hexose in a sample from a normal healthy subject or a population of normal healthy subjects. Any level above the reference level may be deemed to be significantly different from the average level of UDP-hexose in a sample from a normal healthy subject or a population of normal healthy subjects.

The reference level may be at least one standard deviation, at least two standard deviations, or at least three standard deviations above the average level of UDP-hexose in a sample from a subject that has not had kidney failure, AKI, or CKD or a population of subjects that have not had kidney failure, AKI, or CKD. Any level above the reference level may be deemed to be significantly different from the average level of UDP-hexose from a subject who has not had kidney failure, AKI, or CKD or a population of subjects who have not had kidney failure, AKI, or CKD.

The reference level may be at least one standard deviation, at least two standard deviations, or at least three standard deviations above the average level of UDP-hexose in a sample from one or more subjects who have an underlying disease, disorder, or condition, such as diabetes, high blood pressure, low blood pressure, nephrotic syndrome, glomerulonephritis, or polycystic kidney disease, blockage of the urinary tract, muscle breakdown, or hemolytic uremic syndrome, but did not develop kidney failure, AKI, or CKD. Any level above the reference level may be deemed to be significantly different from the average level of UDP-hexose from one or more subjects who have the underlying disease, disorder, or condition but did not develop kidney failure, AKI, or CKD.

The reference level may be a level of UDP-hexose in a control sample, a pooled sample of control individuals, or a numeric value or range of values based on the same. It is also contemplated that a set of standards may be established with reference levels providing thresholds indicative of the severity of renal inflammation.

The reference level may be a level of UDP-hexose in a sample of the same subject measured at an earlier time point. The reference level may be at least one standard deviation, at least two standard deviations, or at least three standard deviations above a level of UDP-hexose in a sample obtained from the same subject at an earlier time point. Any level above the reference level may be deemed to be significantly different from the level in the earlier sample.

In some embodiments, the level of UDP-hexose measured in a sample from a subject identified as having renal inflammation may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, or at least 300% higher than the reference level.

The reference level may be adjusted to account for variables such as sample type, gender, age, weight, and ethnicity. Thus, reference levels accounting for these and other variables may provide added accuracy for the methods described herein.

Computer systems for measuring UDP-hexose levels

In some embodiments of the assays and/or methods described herein, the assay/method comprises or consists essentially of a system for determining (e.g. transforming and measuring) the level of UDP-hexose as described herein and comparing it to a reference level. If the comparison system, which may be a computer implemented system, indicates that the amount of the measured level of UDP-hexose is statistically higher than that of the reference amount, the subject from which the sample is collected may be identified as having renal inflammation. In one embodiment, provided herein is a system comprising: (a) at least one memory containing at least one computer program adapted to control the operation of the computer system to implement a method that includes (i) a determination module configured to measure the level of UDP-hexose in a test sample obtained from a subject; (ii) a storage module configured to store output data from the determination module; (iii) a computing module adapted to identify from the output data whether the measured level of UDP-hexose in the test sample obtained from the subject is higher, by a statistically significant amount, than a reference level, and to provide a retrieved content; (iv) a display module for displaying for retrieved content (e.g., the amount of the measured level of UDP-hexose , or whether the measured level of UDP- hexose is higher than the reference level); and (b) at least one processor for executing the computer program.

Embodiments may be described through functional modules, which are defined by computer executable instructions recorded on computer readable media and which cause a computer to perform method steps when executed. The modules are segregated by function for the sake of clarity. However, it should be understood that the modules/systems need not correspond to discreet blocks of code and the described functions may be carried out by the execution of various code portions stored on various media and executed at various times.

Furthermore, it should be appreciated that the modules may perform other functions, thus the modules are not limited to having any particular functions or set of functions.

The computer-readable storage media may be any available tangible media that can be accessed by a computer. Computer readable storage media includes volatile and nonvolatile, removable and non-removable tangible media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer readable storage media includes, but is not limited to, RAM (random access memory), ROM (read only memory), EPROM (erasable programmable read only memory), EEPROM (electrically erasable programmable read only memory), flash memory or other memory technology, CD-ROM (compact disc read only memory), DVDs (digital versatile disks) or other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and non-volatile memory, and any other tangible medium which can be used to store the desired information and which can accessed by a computer including and any suitable combination of the foregoing. Computer-readable data embodied on one or more computer-readable media may define instructions, for example, as part of one or more programs that, as a result of being executed by a computer, instruct the computer to perform one or more of the functions described herein, and/or various embodiments, variations and combinations thereof. Such instructions may be written in any of a plurality of programming languages, for example, Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic, COBOL assembly language, and the like, or any of a variety of combinations thereof. The computer-readable media on which such instructions are embodied may reside on one or more of the components of either of a system, or a computer readable storage medium described herein, may be distributed across one or more of such components.

The computer-readable media may be transportable such that the instructions stored thereon may be loaded onto any computer resource to implement the aspects of the technology discussed herein. In addition, it should be appreciated that the instructions stored on the computer-readable medium, described above, are not limited to instructions embodied as part of an application program running on a host computer. Rather, the instructions may be embodied as any type of computer code (e.g., software or microcode) that can be employed to program a computer to implement aspects of the technology described herein. The computer executable instructions may be written in a suitable computer language or combination of several languages. Basic computational biology methods are known to those of ordinary skill in the art and are described in, for example, Setubal and Meidanis et ah, Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.),

Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2nd ed., 2001).

The functional modules of certain embodiments may include at minimum a determination module, a storage module, a computing module, and a display module. The functional modules may be executed on one, or multiple, computers, or by using one, or multiple, computer networks. The determination module has computer executable instructions to provide e.g., levels of expression products, etc. in computer readable form.

The determination module may comprise any system for detecting a signal resulting from the detection of UDP-glucose in a biological sample. In some embodiments, such systems may include an instrument, e.g., a plate reader for measuring absorbance. In some embodiments, such systems may include an instrument, e.g., such as the instrument sold under the trade name Cell Biosciences NANOPRO 1000™ System (Protein Simple; Santa Clara, CA), for quantitative measurement of proteins.

The information determined in the determination system may be read by the storage module. As used herein the "storage module" is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the technology described herein include stand-alone computing apparatus, data telecommunications networks, including local area networks (LAN), wide area networks (WAN), Internet, Intranet, and Extranet, and local and distributed computer processing systems. Storage modules also include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage media, magnetic tape, optical storage media such as CD-ROM, DVD, electronic storage media such as RAM, ROM, EPROM, EEPROM and the like, general hard disks and hybrids of these categories such as

magnetic/optical storage media. The storage module is adapted or configured for having recorded thereon, for example, sample name, patient name, and numerical value of the level of ETDP-glucose. Such information may be provided in digital form that may be transmitted and read electronically, e.g., via the Internet, on diskette, via USB (universal serial bus) or via any other suitable mode of communication. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising expression level information.

In one embodiment of any of the systems described herein, the storage module stores the output data from the determination module. In additional embodiments, the storage module stores the reference information such as levels of UDP-hexose in healthy subjects. In some embodiments, the storage module stores the information such as levels of UDP-hexose measured from the same subject in earlier time points.

The computing module may use a variety of available software programs and formats for computing the levels of UDP-hexose. Such algorithms are well established in the art. A skilled artisan is readily able to determine the appropriate algorithms based on the size and quality of the sample and type of data. The data analysis may be implemented in the computing module. In one embodiment, the computing module further comprises a comparison module, which compares the level of UDP-hexose in the test sample obtained from a subject as described herein with the reference level. For example, when the level of UDP-hexose in the test sample obtained from a subject is measured, a comparison module may compare or match the output data, e.g. with the reference level. In certain embodiments, the reference level has been pre-stored in the storage module. During the comparison or matching process, the comparison module may determine whether the level of UDP-hexose in the test sample obtained from a subject is higher than the reference level to a statistically significant degree. In various embodiments, the comparison module may be configured using existing commercially- available or freely-available software for comparison purpose, and may be optimized for particular data comparisons that are conducted.

The computing and/or comparison module, or any other module, may include an operating system (e.g., UNIX) on which runs a relational database management system, a World Wide Web application, and a World Wide Web server. World Wide Web application includes the executable code necessary for generation of database language statements (e.g., Structured Query Language (SQL) statements). Generally, the executables will include embedded SQL statements. In addition, the World Wide Web application may include a configuration file which contains pointers and addresses to the various software entities that comprise the server as well as the various external and internal databases which must be accessed to service user requests. The Configuration file also directs requests for server resources to the appropriate hardware, as may be necessary should the server be distributed over two or more separate computers. In one embodiment, the World Wide Web server supports a TCP/IP protocol. Local networks such as this are sometimes referred to as "Intranets." An advantage of such Intranets is that they allow easy communication with public domain databases residing on the World Wide Web (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in a particular preferred embodiment, users can directly access data (via Hypertext links for example) residing on Internet databases using a HTML interface provided by Web browsers and Web servers.

The computing and/or comparison module provides a computer readable comparison result that can be processed in computer readable form by predefined criteria, or criteria defined by a user, to provide content based in part on the comparison result that may be stored and output as requested by a user using an output module, e.g., a display module.

In some embodiments, the content displayed on the display module may be the relative levels of UDP-hexose in the test sample obtained from a subject as compared to a reference level. In certain embodiments, the content displayed on the display module may indicate whether the levels of UDP-hexose are found to be statistically significantly higher in the test sample obtained from a subject as compared to a reference level. In some embodiments, the content displayed on the display module may show the levels of UDP-hexose from the subject measured at multiple time points, e.g., in the form of a graph. In some embodiments, the content displayed on the display module may indicate whether the subject has renal inflammation. In certain embodiments, the content displayed on the display module may indicate whether the subject is in need of a treatment for renal inflammation.

In one embodiment, the content based on the computing and/or comparison result is displayed on a computer monitor. In one embodiment, the content based on the computing and/or comparison result is displayed through printable media. The display module may be any suitable device configured to receive from a computer and display computer readable information to a user. Non-limiting examples include, for example, general-purpose computers such as those based on Intel PENTIUM-type processor, Motorola PowerPC, Sun UltraSPARC, Hewlett- Packard PA-RISC processors, any of a variety of processors available from Advanced Micro Devices (AMD) of Sunnyvale, California, or any other type of processor, visual display devices such as flat panel displays, cathode ray tubes and the like, as well as computer printers of various types.

In one embodiment, a World Wide Web browser is used for providing a user interface for display of the content based on the computing/comparison result. It should be understood that other modules may be adapted to have a web browser interface. Through the Web browser, a user can construct requests for retrieving data from the computing/comparison module. Thus, the user will typically point and click to user interface elements such as buttons, pull down menus, scroll bars and the like conventionally employed in graphical user interfaces.

Systems and computer readable media described herein are merely illustrative

embodiments of the technology relating to determining the levels of UDP-hexose, and therefore are not intended to limit the scope of the invention. Variations of the systems and computer readable media described herein are possible and are intended to fall within the scope of the invention. The modules of the machine, or those used in the computer readable medium, may assume numerous configurations. For example, function may be provided on a single machine or distributed over multiple machines.

Improvement of renal function

Providing a reagent that decreases the availability of UDP-hexoses to bind to P2Y 14 may improve renal function. For example, renal function may be improved by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, or at least 300%. Measurable markers of renal function, are well known in the medical and veterinary literature and to those of skill in the art, and include, but are not limited to, blood urea nitrogen or "BUN" levels (both static measurements and measurements of rates of increase or decrease in BUN levels), serum creatinine levels (both static measurements and measurements of rates of increase or decrease in serum creatinine levels), measurements of the BUN/creatinine ratio (static measurements of measurements of the rate of change of the BUN/creatinine ratio), urine/plasma ratios for creatinine, urine/plasma ratios for urea, glomerular filtration rates (GFR), serum concentrations of sodium (Na+) or potassium (K+), urine osmolarity, daily urine output, urine protein/creatinine ratio, albuminuria, and the like. Of the above, measurements of the plasma concentrations of creatinine and/or urea or BUN are particularly important and useful readouts of renal function.

Incorporation by Reference

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

Equivalents

Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.