Compositions and methods for treating and preventing kidney disease

Abstract

Provided herein are compositions and methods for treating and preventing kidney disease. In particular, provided herein are methods of treating and preventing kidney disease with SGLT2 inhibitors in a subject with low levels of urinary EGF.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING AND PREVENTING KIDNEY

[0002] DISEASE

[0003] STATEMENT OF RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 529,098, filed July 26, 2023, the entire contents of which are incorporated herein by reference for all purposes.

[0005] FIELD OF THE DISCLOSURE

[0006] Provided herein are compositions and methods for treating and preventing kidney disease. In particular, provided herein are methods of treating and preventing kidney disease with SGLT2 inhibitors in a subject with low levels of urinary EGF.

[0007] BACKGROUND

[0008] Chronic kidney disease, also called chronic kidney failure, involves a gradual loss of kidney function. Advanced chronic kidney disease can cause dangerous levels of fluid, electrolytes and wastes to build up in the body.

[0009] Diseases and conditions that cause or increase the risk of developing chronic kidney disease include type 1 or type 2 diabetes, high blood pressure, glomerulonephritis, interstitial nephritis, polycystic kidney disease or other inherited kidney diseases, prolonged obstruction of the urinary tract (e.g., from conditions such as enlarged prostate, kidney stones and some cancers), vesicoureteral reflux, and recurrent kidney infection.

[0010] Treatment for chronic kidney disease focuses on slowing the progression of kidney damage, usually by controlling the cause. Controlling the cause does not always keep kidney damage from progressing. Chronic kidney disease can progress to end-stage kidney failure, which is fatal without artificial filtering (dialysis) or a kidney transplant.

[0011] Additional treatments for kidney disease are needed.

[0012] BRIEF SUMMARY OF THE DISCLOSURE

[0013] Provided herein are compositions and methods for treating and preventing kidney disease. In particular, provided herein are methods of treating and preventing kidney disease with SGLT2 inhibitors in a subject with low levels of urinary EGF.

[0014] Experiments described herein demonstrated that decreased uEGF was associated with increased kidney disease progression in patients. Use of SGLT2 inhibitors was shown to increase kidney tissue expression of EGF (reflected by increased urinary EGF) and was associated with a downstream signaling cascade linked to tubular repair and reversal of tubular injury.

[0015] Accordingly, in some embodiments, provided herein is a method of treating or preventing kidney disease, comprising: a) assaying a urine sample from a subject diagnosed with kidney disease for the level of urine epidermal growth factor (uEGF); and b) administering an SGLT2 inhibitor to a subject with a level of uEGF below a threshold level (e.g., less than 10 ng / mg creatinine (e.g., less than 9 ng / mg, 8ng / mg, 7 ng / mg, 6 ng / mg, 5 ng / mg, 4 ng / mg, 3 ng / mg, or 2 ng / mg; normalized by urinary creatinine levels to adjust for hydration status)).

[0016] Further provided is a method of treating or preventing kidney disease, comprising: a) administering an SGLT2 inhibitor to a subject with kidney disease; and b) assaying a urine sample from the subject for the level of uEGF (e.g., before and after treatment). In some embodiments, the level of uEGF and / or the change in concentration of uEGF is used to determine a treatment (e.g., start, stop, or change a treatment). For example, in some embodiments, if uEGF is increasing or above a threshold, indicating a response to the treatment, the treatment is continued. In some embodiments, a decrease in uEGF or uEGF below a threshold level is indicative of the need for an alternative treatment (e.g., a different SGLT2 inhibitor or a treatment that is not a SGLT2 inhibitor).

[0017] Also provided is a method of providing a prognosis to a subject with kidney disease or at risk of kidney disease, comprising: a) assaying a urine sample from a subject diagnosed with kidney disease for the level of uEGF; and b) providing a prognosis of increased risk of severe disease when the level of uEGF is below a threshold level.

[0018] Additional embodiments provide a method of recommending a treatment course of action in a subject with kidney disease or at risk of kidney disease, comprising: a) assaying a urine sample from a subject diagnosed with kidney disease for the level of uEGF; and b) recommending treatment with a SGLT2 inhibitor when the level of uEGF is below a predetermined level.

[0019] In some embodiments, the level of uEGF is measured as a uEGF to creatinine ratio (uEGF / Cr). In some embodiments, the threshold level is predetermined. In some embodiments, the subject has type II diabetes. In some embodiments, the administering increases the level of EGF in the kidneys of the subject.

[0020] The present disclosure is not limited to particular SGLT2 inhibitors. Examples include but are not limited to, canagliflozin, dapagliflozin, empagliflozin, or ertugliflozin. In some embodiments, the administering is at regular intervals (e.g., one or more times a day, weekly, monthly, etc.) for a period of time (e.g., weeks, months, years, or indefinitely). In some embodiments, the assaying is repeated one or more times during and / or after the administering (e.g., at regular or irregular intervals such as, once every week, month, or one or more times per year).

[0021] In yet other embodiments, provided herein is the use of an SGLT2 inhibitor to treat or prevent kidney disease in a subject with a level of uEGF below a threshold level.

[0022] Additional embodiments are described herein.

[0023] BRIEF DESCRIPTION OF THE FIGURES

[0024] FIG. 1 shows associations of baseline uEGF / Cr with the composite kidney outcome by subpopulations defined by treatment allocation, age, sex, UACR, eGFR, and cardiovascular disease history. Hazard ratios are expressed per doubling of uEGF / Cr.

[0025] FIG. 2 shows a forest plot of the unadjusted effect of canagliflozin on the composite kidney outcome by tertiles of baseline uEGF / Cr.

[0026] FIG. 3 shows ScRNAseq analysis showing restricted expression of EGF mRNA in TAL and DCT cells, a Unsupervised clustering of cells from reference tissue from healthy participants and patients with T2D with / without SGLT2i treatment, b For all groups [HC, T2Di(-) and T2Di(+)], EGF mRNA expressing cells (green dots) were limited to the TAL and DCT clusters, c Dot plots analysis of EGF expression in TAL cell cluster from kidney biopsies of healthy participants (HC:TAL), young persons with T2D (T2Di (-)_TAL), and young persons with T2D treated with SGLT2i (T2Di(+)_TAL).

[0027] FIG. 5 shows a flow diagram of available samples for measurement.

[0028] FIG. 6 shows a Pearson correlation test of baseline uEGF / Cr with covariates used in the assessment of the association of baseline uEGF / Cr with the composite kidney outcome.

[0029] FIG. 7 shows a Pearson correlation test of the 1 -year change in uEGF / Cr with covariates used in the assessment of the association of the 1-year change in uEGF / Cr from baseline with the composite kidney outcome.

[0030] FIG. 8 shows UMAP demonstration of annotated cell clusters corresponding to major cell types in the nephron in three biopsy groups: HC (n=6), T2Di (-) (n=6), and T2Di(+) (n=10). B. Charts displaying each cell cluster had a robust representation in the 3 biopsy groups.

[0031] FIG. 9 shows Co-localization of EGF with TAL-specific marker gene UMOD and DCT-specific gene SLC12A3. A) Cell specific markers used to define cell clusters. Red and blue boxes / arrows indicate UMOD and SCL12A3 are used to define the TAL and DCT clusters, respectively. B) and C) UMAP presentations demonstrate the expression of EGF (green), UMOD (red in B), and SLC12A3 (red in C). Yellow areas indicate the co-locaization of the EGF with these two cell lineage specific genes respectively.

[0032] FIG. 10 shows a hierarchical summary of significantly enriched pathways / networks and upstream regulators (cytokines, growth factors and transcriptional factors) in EGF coexpressing gene signatures in TALs of patients with / without SGLT2i treatment. EDN1 as the top node of the EGF co-regulated gene signature in TAL cells of DKD patients without SGLT2i (upper panel), whereas in patients treated with SGLT2i, EGF is the top node impacting enriched pathways / networks (lower panel). Light orange nodes represent up- regulated / activated genes / pathways whereas light blue nodes represent down- regulated / repressed genes / pathways .

[0033] DETAILED DESCRIPTION OF THE DISCLOSURE

[0034] Provided herein are compositions and methods for treating and preventing kidney disease. In particular, provided herein are methods of treating and preventing kidney disease with SGLT2 inhibitors in a subject with low levels of urinary EGF.

[0035] Definitions

[0036] The term "chronic kidney disease" (CKD) refers to a condition defined as abnormalities of kidney structure or function, with implications for health, which can occur abruptly, and either resolve or become chronic (Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease Guidelines (KDIGO 2012). CKD is a general term for heterogeneous disorders affecting kidney structure and function with variable clinical presentation, in part related to cause, severity and the rate of progression (Kidney International Supplements (2013) 3, vii). CKD is usually an irreversible and progressive disease and can lead to kidney failure, also called End Stage Renal Disease (ESRD) or End Stage Kidney Disease (ESKD), over time if it is not treated.

[0037] As used herein, the terms “detect,” “detecting,” or “detection” may describe either the general act of discovering or discerning or the specific observation of a composition. Detecting a composition may comprise determining the presence or absence of a composition. Detecting may comprise quantifying a composition. For example, detecting comprises determining the expression level of a composition. The composition may comprise a nucleic acid molecule or a protein or peptide. For example, the composition may comprise at least a portion of EGF. Alternatively, or additionally, the composition may be a detectably labeled composition. Alternatively, or additionally, the composition may be a detectably labeled composition.

[0038] As used herein, the term “subject” refers to any organisms that are treated or screened using the diagnostic methods described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans.

[0039] The term “diagnosed,” as used herein, refers to the recognition of a disease by its signs and symptoms, or genetic analysis, pathological analysis, histological analysis, and the like.

[0040] The term "label" as used herein refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect, and that can be attached to a nucleic acid or protein. Labels include but are not limited to dyes; radiolabels such as32P; binding moieties such as biotin; haptens such as digoxgenin; luminogenic, phosphorescent or Anorogenic moieties; and Auorescent dyes alone or in combination with moieties that can suppress or shift emission spectra by Auorescence resonance energy transfer (FRET). Labels may provide signals detectable by Auorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. A label may be a charged moiety (positive or negative charge) or alternatively, may be charge neutral. Labels can include or consist of nucleic acid or protein sequence, so long as the sequence comprising the label is detectable.

[0041] As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass Auids (e.g., blood, urine), solids, tissues, and gases. Biological samples include urine, urine supernatant, and urine cell pellet as well as blood products, such as plasma, serum and the like. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure.

[0042] Alustrative Embodiments

[0043] Provided herein are compositions and methods for treating and preventing kidney disease. In particular, provided herein are methods of treating and preventing kidney disease with SGLT2 inhibitors in a subject with low levels of urinary EGF.

[0044] Sodium-glucose cotransporter 2 (SGLT2) inhibitors block reabsorption of sodium and glucose in the proximal tubule. Epidermal growth factor (EGF) is a key mitogenic factor involved in cell proliferation, hypertrophy migration, and differentiation of epithelial cells (6,7). EGF is predominantly produced in the ascending loop of Henle and distal convoluted tubule (DCT) and exerts its effect through binding to the EGF receptor, which is widely expressed in the kidney along the glomerulus, loop of Henle, DCT, and collecting duct (7). Decreased excretion of urinary EGF (uEGF) is present in different kidney pathologies, including patients with T2D and CKD (8).

[0045] Experiments described herein determined that uEGF levels (e.g., baseline uEGF normalized by urine creatinine (uEGF / Cr)) are associated with adverse kidney outcomes (e.g., sustained 40% decline in eGFR, kidney failure, kidney death, reduced eGFR, increased UACR, CKD stage advance, etc.) (e.g., in patients with T2D at high cardiovascular risk). Further experiments determined that SGLT2 inhibitors increased the concentration of uEGF. Additionally, single-cell RNA sequencing from kidney biopsies from young persons with T2D who were and were not using SGLT2 inhibitors from the two clinical studies was used to assess gene expression network changes related to SGLT2 inhibition.

[0046] In some embodiments, the present disclosure provides improved treatments for kidney disease by identifying subjects for treatment with SGLT2 inhibitors, monitoring treatment with SGLT2 inhibitors, and providing prognostic information to subjects with kidney disease.

[0047] Methods of detecting uEGF uEGF proteins can be detected using any suitable method. In some embodiments, the level of uEGF is quantified as a uEGF to creatinine ratio (uEGF / Cr), although other normalization methods or factors may be used.

[0048] In some embodiments, the patient sample is subjected to preliminary processing designed to isolate or enrich the sample for EGF. A variety of techniques known to those of ordinary skill in the art may be used for this purpose, including but not limited to: centrifugation, immunocapture, and cell lysis.

[0049] Samples are assayed for uEGF protein using suitable methods such as Westerns and immunoassays, like ELISAs, RIAs, and fluorescence-based immunoassays. Further suitable methods to detect EGF include measuring a physical or chemical property specific for the peptide or polypeptide such as its precise molecular mass or NMR spectrum. Said methods comprise, e.g., biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass- spectrometers, NMR- analyzers, or chromatography devices. Further, methods include microplate ELISA-based methods, fully automated or robotic immunoassays, and latex agglutination assays. Illustrative non-limiting examples of immunoassays include but are not limited to: immunoprecipitation; Western blot; ELISA; immunohistochemistry; immunocytochemistry; flow cytometry; and immuno-PCR. Polyclonal or monoclonal antibodies delectably labeled using various techniques known to those of ordinary skill in the art (e.g., colorimetric, fluorescent, chemiluminescent or radioactive) are suitable for use in the immunoassays. Immunoprecipitation is the technique of precipitating an antigen out of solution using an antibody specific to that antigen. The process can be used to identify protein complexes present in cell extracts by targeting a protein believed to be in the complex. The complexes are brought out of solution by insoluble antibody -binding proteins isolated initially from bacteria, such as Protein A and Protein G. The antibodies can also be coupled to sepharose beads that can easily be isolated out of solution. After washing, the precipitate can be analyzed using mass spectrometry, Western blotting, or any number of other methods for identifying constituents in the complex.

[0050] A Western blot, or immunoblot, is a method to detect protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate denatured proteins by mass. The proteins are then transferred out of the gel and onto a membrane, typically polyvinyldiflroride or nitrocellulose, where they are probed using antibodies specific to the protein of interest. As a result, researchers can examine the amount of protein in a given sample and compare levels between several groups.

[0051] An ELISA, short for Enzyme- Linked ImmunoSorbent Assay, is a biochemical technique to detect the presence of an antibody or an antigen in a sample. It utilizes a minimum of two antibodies, one of which is specific to the antigen and the other of which is coupled to an enzyme. The second antibody will cause a chromogenic or fluorogenic substrate to produce a signal. Variations of ELISA include sandwich ELISA, competitive ELISA, and ELISPOT. Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations and also for detecting the presence of antigen.

[0052] Immunohistochemistry and immunocytochemistry refer to the process of localizing proteins in a tissue section or cell, respectively, via the principle of antigens in tissue or cells binding to their respective antibodies. Visualization is enabled by tagging the antibody with color producing or fluorescent tags. Typical examples of color tags include, but are not limited to, horseradish peroxidase and alkaline phosphatase. Typical examples of fluorophore tags include, but are not limited to, fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Immuno-polymerase chain reaction (IPCR) utilizes nucleic acid amplification techniques to increase signal generation in antibody-based immunoassays. Because no protein equivalence of PCR exists, that is, proteins cannot be replicated in the same manner that nucleic acid is replicated during PCR, the only way to increase detection sensitivity is by signal amplification. The target proteins are bound to antibodies which are directly or indirectly conjugated to oligonucleotides. Unbound antibodies are washed away, and the remaining bound antibodies have their oligonucleotides amplified. Protein detection occurs via detection of amplified oligonucleotides using standard nucleic acid detection methods, including real-time methods.

[0053] Any suitable EGF antibody may be utilized in detection methods described herein. In some embodiments, commercially available antibodies are utilized (e.g., available from Sigma Aldrich (St. Lous, MS), Abeam (Cambridge, EK), or Novus Biologicals (Centennial CO)).

[0054] Threshold levels

[0055] In some embodiments, threshold levels of uEGF are used to provide or recommend treatments or prognoses to a subject. Certain embodiments utilize a predetermined threshold level. In some embodiments, the threshold level is a level of uEGF in healthy individuals or populations. In some embodiments, the threshold level is the level of uEGF in individuals or populations with kidney disease or type II diabetes that have been shown to respond to a particular treatment (e.g., SGLT2 inhibitor). References levels may represent population averages or alternatively, be based on a baseline level of an individual (e.g., before treatment or during treatment). In some embodiments, the threshold level is patient-specific and changes in a patient’s threshold level are monitored over time.

[0056] The threshold level may vary depending on various physiological parameters such as age, gender or subpopulation, as well as on the means used for the determination of the uEGF level. In one embodiment, the reference sample is from essentially the same type of cells, tissue, organ or body fluid source as the sample from the individual or patient subjected to the method of the disclosure, e.g., if urine is used as a sample to determine the level uEGF in the individual, the threshold level is also determined in urine or a part thereof.

[0057] In certain embodiments, the term “at the threshold level” refers to a level of the uEGF in the sample from the individual or patient that is essentially identical to the threshold level or to a level that differs from the threshold level by up to 1%, up to 2%, up to 3%, up to 4%, up to 5%. In certain embodiments, the term “decrease” or “below” herein refers to a level of uEGF in the sample from the individual or patient below the threshold level or to an overall reduction of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater, determined by the methods described herein, as compared to the threshold level.

[0058] In certain embodiments, the term “increase” or “above” herein refers to a level of uEGF in the sample from the individual or patient above threshold level or to an overall increase of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater, determined by the methods described herein, as compared to the threshold level.

[0059] In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of uEGF) into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. Thus, in some preferred embodiments, the present disclosure provides the further benefit that the clinician, who is not likely to be trained in genetics or molecular biology, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.

[0060] The present disclosure contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, information providers, medical personnel, and subjects. For example, in some embodiments of the present disclosure, a sample (e.g., a urine sample) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g., a urine sample) and directly send it to a profiling center. Where the sample comprises previously determined biological information, the information may be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems). Once received by the profiling service, the sample is processed and a profile is produced (i.e., uEGF level), specific for the diagnostic or prognostic information desired for the subject. The profile data is then prepared in a format suitable for interpretation by one or more medical personnel (e.g., a treating clinician, physician assistant, nurse, or pharmacist). For example, rather than providing raw expression data, the prepared format may represent a diagnosis or risk assessment (e.g., risk of developing severe kidney disease or likelihood to respond to a treatment) for the subject, along with recommendations for particular treatment options. The data may be displayed to the medical personnel by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed for the medical personnel (e.g., at the point of care) or displayed to the medical personnel on a computer monitor.

[0061] In some embodiments, the information is first analysed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and / or to convert the raw data to information useful for medical personnel or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the medical personnel, the subject, or researchers.

[0062] In some embodiments, the subject is able to directly access the data using the electronic communication system. The subject may choose further intervention or counselling based on the results.

[0063] Methods

[0064] Certain embodiments of the disclosure provide methods of providing a prognosis, recommending a treatment course of action, or treating a subject with kidney disease.

[0065] For example, in some embodiments, uEGF levels are used to providing a prognosis to a subject with kidney disease or at risk of kidney disease (e.g., to provide a prognosis of increased risk of severe disease when the level of uEGF is below a threshold level).

[0066] In some embodiments, methods of recommending a treatment are provided. For example, in some embodiments, treatment with a SGLT2 inhibitor is recommended when the level of uEGF is below a threshold level.

[0067] In some embodiments, provided herein is a method of treating or preventing kidney disease, comprising: a) assaying a urine sample from a subject diagnosed with kidney disease for the level of uEGF; and b) administering an SGLT2 inhibitor to a subject with a level of uEGF below a threshold level. The present disclosure is not limited to a particular threshold level of uEGF. In some embodiments, the threshold level is a value less than 50 ng / ml (e.g., less than 40 ng / mg, less than 30 ng / mg, less than 20 ng / mg, less than 15 ng / mg, less than 10 ng / mg, less than 9 ng / mg, 8ng / mg, 7 ng / mg, 6 ng / mg, 5 ng / mg, 4 ng / mg, 3 ng / mg, or 2 ng / mg; normalized by urinary creatinine levels to adjust for hydration status).

[0068] In some embodiments, levels of uEGF are monitored before and / or during treatment in order to determine the efficacy of the treatment. For example, if levels of uEGF increase, treatment with the SGLT inhibitor is continued. If level remain low or decrease, alternative treatments are considered.

[0069] Levels of uEGF are monitored at any suitable regular or irregular interval (e.g., daily, weekly, monthly, or one or more times per year).

[0070] In some embodiments, the subject has type II diabetes or another condition that increases the risk of kidney disease or severe kidney disease.

[0071] In some embodiments, the administering increases the level of EGF in the kidneys of said subject.

[0072] The present disclosure is not limited to particular SGLT2 inhibitors. Examples include but are not limited to, canagliflozin, dapagliflozin, empagliflozin, or ertugliflozin.

[0073] Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual SGLT2 inhibitors, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 pg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.

[0074] In some embodiments, canagliflozin is given at a dosage of 50 to 400 mg (e.g., 100 to 300 or 100 mg) per day. In some embodiments, dapagliflozin is given at a dosage of 2.5 to 20 mg (e.g., 5 to 10 mg) per day. In some embodiments, empagliflozin is given at a dosage of 5 to 40 mg (e.g., 10 to 25 mg) per day. In some embodiments, ertugliflozin is given at a dosage of 2.5 to 25 mg (e.g., 5 to 15 mg) per day. In some embodiments, the administering is at regular intervals (e.g., one or more times a day, weekly, monthly, etc.) for a period of time (e.g., weeks, months, years, or indefinitely). In some embodiments, the assaying is repeated one or more times during and / or after said administering (e.g., at regular or irregular intervals such as, once every week, month, or one or more times per year).

[0075] In some embodiments, one or more additional treatments are administered in combination with the SGLT2 inhibitor. Examples include but are not limited to, agents of the class of angiotensin converting enzyme inhibitors (ACEi) or of the class of the angiotensin receptor blockers (ARBs). Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a dose may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by exigencies of the therapeutic situation.

[0076] EXPERIMENTAL

[0077] The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present disclosure and are not to be construed as limiting the scope thereof.

[0078] Methods

[0079] Patients and study design of the CANVAS trial

[0080] The CANVAS trial was a multicenter, double-blinded, placebo-controlled, randomized clinical trial that assessed the safety and efficacy of the SGLT2 inhibitor canagliflozin on cardiovascular and kidney outcomes in participants with T2D at high cardiovascular risk or who had a history of cardiovascular disease. Results of this trial have been reported previously (2). In brief, the CANVAS trial enrolled 4330 participants from 24 countries. Participants were randomized to 100 mg or 300 mg canagliflozin or matching placebo in a 1 :1 :1 ratio. The median follow-up duration was 6.1 years. Before trial initiation, all participants were offered the possibility to participate in the exploratory biomarker research study, where blood and urine samples of the participants were stored for future biomarker exploratory research. All participants, care providers, trial staff, and outcome assessors were blinded to treatment allocation during the study. Written informed consent was obtained before study initiation. The informed consent for blood and urine collection for biomarker research was separate and optional. The CANVAS trial was conducted following the principles of the Declaration of Helsinki and was registered with clinicaltrials.gov (NCT01032629). The CANVAS trial was approved by an ethics committee at each participating site.

[0081] Inclusion criteria for the CANVAS trial were a diagnosis of T2D with an HbAiclevel of >58 mmol / mol (7.0%) and <91 mmol / mol (10.5%) and an age of 30 years or older and a history of symptomatic atherosclerotic cardiovascular disease history, or an age of 50 years or older with two or more risk factors for cardiovascular disease history. These risk factors included a duration of type 2 diabetes of at least 10 years, systolic BP >140 mmHg, treatment with >1 antihypertensive agent, current smoking status, diagnosis of micro- or macroalbuminuria, and HDL cholesterol level of <1 mmol / 1. At inclusion patients also needed to meet other criteria for inclusion including an eGFR of >30 min / min / 1.73m2. The appendix of the first publication of the CANVAS trial includes the full list of these criteria.

[0082] Patients and study design of the biopsy study for EGF scRNA-Seq analysis

[0083] Adolescents and young adults (N=16) with youth-onset T2D (12-21 years of age, type 2 diabetes onset <18 years of age, diabetes duration 1-10 years and HbAic <11%) from the Renal Hemodynamics, Energetics and Insulin Resistance in Youth Onset Type 2 Diabetes Study (Renal-HEIR, NCT03584217) and the Impact of Metabolic Surgery on Pancreatic, Renal and Cardiovascular Health in Youth with Type 2 Diabetes (IMPROVE- T2D, NCT03620773) who underwent an optional research kidney biopsy were included in this analysis. These participants were recruited from the Type 2 Diabetes and Metabolic Bariatric Surgery clinics at the Children’s Hospital Colorado at the Anschutz Medical Campus in Aurora, Colorado. T2D was defined by the American Diabetes Association criteria plus the absence of glutamic acid decarboxylase, islet cell, zinc transporter 8 and / or insulin autoantibodies. Main exclusion criteria for the optional kidney biopsy included evidence of a bleeding disorder or complications from bleeding, use of aspirin, NSAIDs or other blood thinners that could not be safely stopped for a sufficient time period before and after the biopsy, eGFR < 40 ml / min / 1.73m2, single kidney (either by history, documented by prior imaging or ultrasound performed prior to the biopsy), and uncontrolled or difficult to control hypertension (> 150 / 90 mm Hg at day of biopsy).

[0084] The Renal-HEIR and IMPROVE-T2D cohorts have intentionally harmonized study protocols and were both approved by the Colorado Multiple Institutional Review Board (COMIRB). Participants and / or parents provided written informed assent and / or consent as appropriate for age. Participants who opted to undergo the optional kidney biopsy were specifically and additionally consented by the research and biopsy teams. Medication use was recorded for all participants, and type 2 diabetes treatment was prescribed at the discretion of their medical provider. Normative reference tissue was provided by 6 healthy adult participants in the Control of Renal Oxygen Consumption, Mitochondrial Dysfunction, and Insulin Resistance (CROCODILE) study (NCT04074668).

[0085] Urinary EGF assay and single cell RNA sequence assessment in kidney biopsies

[0086] Urine samples for exploratory biomarker research were collected and stored at baseline and week 52 after randomization. For this study the Mesoscale Quickplex SQ 120 platform (Meso Scale Diagnostics [MSD], Rokcville, MD, USA), which is a high- performance electrochemiluminescence immunoassay, was used to measure EGF in urine at baseline and week 52. All urine samples were measured between April 2019 and February 2020. In total, 390 of the 3521 urine samples were measured in duplicate. The mean (minimum, maximum) CV of the duplicates was 5% (0%, 23%).

[0087] To define the RNA expression and regulation patterns of EGF upon SGLT2 inhibitor treatment, scRNA-Seq analysis was performed on cell populations obtained from kidney tissue samples of 10 patients treated with an SGLT2 inhibitor (nine patients were using canagliflozin and one patient empagliflozin), 6 patients under standard care, and 6 healthy reference tissues. Tissue processing, single-cell isolation, and scRNA-Seq data generation were performed according to the protocol developed for the Kidney Precision Medicine Project (KPMP) (10-13).

[0088] Outcomes

[0089] CANVAS

[0090] The composite kidney outcome was defined as a sustained 40% decline of eGFR, endstage kidney disease defined as an eGFR <15 mL / min / 1.73m2, need for dialysis or kidney transplantation, or death related to kidney disease. The kidney outcome was adjudicated by a blinded adjudication committee using predefined and rigorous endpoint definitions.

[0091] Statistical analysis

[0092] CANVAS clinical trial

[0093] Normal distributed continuous variables were reported as means with SDs. Skewed distributed continuous variables were reported as median values with IQRs and underwent natural logarithmic transformation before analyses. Categorical variables were reported as percentages. The hazard ratios for the composite kidney outcome for uEGF / Cr categorized into quartiles or doubling of uEGF / Cr were estimated using multivariable Cox proportional hazard regression. These were estimated by four consecutive models each built with different covariates to assess the impact of the covariates between uEGF / Cr and the composite kidney outcome. The first model was built with age, sex, race, and treatment allocation as covariates. To the second model the covariates HbAlc, systolic blood pressure, body mass index, LDL, HDL and the history of cardiovascular disease were added. The third model included baseline eGFR. Lastly, log transformed UACR was added to the fourth model. The fully adjusted model was also used to explore the association between uEGF and kidney outcomes in subgroups defined by treatment allocation, baseline age, sex, eGFR, UACR, and cardiovascular disease history to assess possible effect modification by these variables.

[0094] It was assessed whether baseline uEGF / Cr modified treatment effect of canagliflozin vs placebo on the composite kidney outcome by fitting Cox proportional hazard regression models. Heterogeneity was tested by adding an interaction term between uEGF / Cr, fitted as categorical variable into tertiles and treatment allocation to the relevant Cox model.

[0095] The placebo corrected effect of canagliflozin on uEGF / Cr was calculated by using an ANCOVA model with the change in uEGF / Cr defined as the outcome and treatment allocation and baseline uEGF / Cr as the covariates. This effect was also assessed for the subgroups defined by baseline UACR, eGFR and markers of inflammation: urinary monocyte chemotactic protein 1 (MCP-1) and Kidney Injury Molecule 1 (KIM-1).

[0096] The association of the 1-year change in uEGF / Cr from baseline with subsequent kidney outcomes was assessed using a Cox proportional hazard regression model adopted to a landmark approach. Any kidney outcomes that occurred in the first year were excluded from the analysis. The 1-year change in uEGF / Cr was categorized into quartiles which were then fitted in Cox proportional hazard regression models. The second quartile was taken as reference, since this was the closest change in uEGF / Cr to zero which was not an increase in uEGF / Cr, to assess the relative hazard ratios of an increase with a decrease or no change. The first model included baseline uEGF / Cr, age, sex, race, and treatment allocation. The baseline and 1-year change in eGFR were added to the second model. The baseline and 1-year change in eGFR with UACR in the third model. The fourth model included all aforementioned covariates. Lastly, history of cardiovascular disease, HbAlc, systolic blood pressure, body mass index, LDL, HDL, and the 1-year change in systolic blood pressure, body mass index, and HbAlc were added. Identification ofEGF coregulated gene signatures and pathway identification.

[0097] ScRNASeq analysis was performed using the cortex region of the kidney biopsy and processed according to the Kidney precision medicine project (KPMP) single cell protocol (1). To summarize, single cells isolated from frozen tissues using Liberase TL were processed by University of Michigan Advanced Genomics Core facility. Standard processing of sample demultiplexing, barcode processing and quantification were performed with 10X Cell Ranger v6 pipeline (2-4). Individual sample matrices were combined using RunHarmony and ambient mRNA content removal using SoupX (Seurat , version 4.0.0) (5-7). Cluster annotation followed the literature derived kidney markers from previous KPMP publications (6-7). Additional QC metrics (6) resulted in cells with gene counts between 500 and 5000, and <50% mitochondrial genes.

[0098] Genes that were differentially expressed in EGF-expressing (EGF+) versus nonexpressing (EGF-) thick ascending loop (TAL) cells from patients with youth-onset T2D with or without SGLT2i treatment were identified using the FindMarkers Seurat function. EGF+ versus EGF- cells were based on greater than 0 (EGF >0) normalized gene expression. For the differential gene signature, Welch t-test was used to compare the differences and genes with Bonferroni-adjusted P-values <1‘20that also had a fold change >1.20 or <-0.83 were selected.

[0099] To determine the significantly enriched biological processes and pathways in the differentially expressed gene sets, the method described previously15and projected gene signatures that were dysregulated in patients with youth-onset T2D and reversed by SGLT2i treatment into the HumanBase functional network were used. Community clustering of the network was used to identify tightly connected sets of genes using the HumanBase module detection function.16Canonical pathway enrichment, upstream regulator and network analysis using Ingenuity Pathway Analysis (IPA, Qiagen, Redwood City, CA, USA) was conducted and a graphic summary of these analytical results was generated.

[0100] Results were deemed significant when P < 0.05 except otherwise stated. All analyses were performed in SAS version 9.4 (SAS Institute, Cary, NC, USA) and Stata version 16.1 (StataCorp, College Station, TX, USA).

[0101] Results

[0102] CANVAS Study population

[0103] Out of the 4330 included participants in the CANVAS trial, 3521 (81.3%) had available urine samples to measure uEGF and determine uEGF / Cr at baseline and 2722 (62.9%) at baseline and 52 weeks follow-up. Out of the 2722 participants, 15 were excluded from the analysis of the association of 1 year change in uEGF / Cr with the composite kidney outcome, since these participants experienced the composite kidney outcome during the first year of follow-up. Baseline characteristics of the 3521 participants are shown in Table 1. The characteristics of the groups allocated to treatment with canagliflozin compared to placebo were well balanced and were similar to the baseline characteristics for the overall CANVAS trial population reported previously. Mean uEGF and uEGR / Cr were 4570 ng / mg and 5.12 pg / g, respectively. Baseline characteristics in quartiles of baseline uEGF / Cr are presented in Table 6.

[0104] Association of uEGFR / Cr with the composite kidney outcome

[0105] The median follow-up duration of the 3521 participants was 6.1 years (IQR 5.8, 6.4), during which 134 participants experienced the composite kidney outcome. In general, Pearson correlations between uEGF / Cr and other covariates were modest to weak, where eGFR was stronger correlated relative to other covariates (Figure 6). In longitudinal analyses, baseline uEGF / Cr was significantly associated with the composite kidney outcome with a corresponding hazard ratio (HR) per doubling uEGF / Cr in the fully adjusted model of 0.88 (95% CI 0.78, 0.99; p=0.04; Table 2). Categorical assessment of the association between uEGF / Cr and the composite kidney outcome revealed that participants in the two upper quartiles of the uEGF / Cr distribution had a statistically significant 2-fold lower risk for the composite kidney outcome when compared to the first quartile (Table 2). Further assessments of the association in subgroups defined by baseline patient characteristics showed no significant difference across these subgroups (Figure 1).

[0106] Effect of canagliflozin on the composite kidney outcome by baseline uEGF / Cr level

[0107] Compared to placebo, canagliflozin reduced the composite kidney outcome in the overall population by 41% (HR 0.59; 95% CI 0.42, 0.83; p<0.002). Fitting uEGF / Cr as continuous or categorical variable showed no evidence that the effect of canagliflozin on the composite kidney outcome varied by baseline uEGF / Cr level (both p values for heterogeneity >0.20; Figure 2).

[0108] Effect of canagliflozin on uEGF / Cr

[0109] Overall, the geometric mean of uEGF / Cr decreased after 52 weeks from baseline in the placebo group while the geometric uEGF / Cr modestly increased after 52 weeks treatment of canagliflozin (Table 3 and Figure 3). Overall, compared to placebo, the mean increase with canagliflozin was 7.3% (95% CI 2.0, 12.8; p=0.01 ). This effect of canagliflozin was consistent in key patient subgroups (Table 3).

[0110] Association of the 1-year change in uEGF / Cr with the composite kidney outcome

[0111] The median uEGF / Cr mean of the 2707 participants with available uEGF / Cr at baseline and week 52 after randomization was 5.25 ng / mg. Among these participants 110 (4.1%) experienced the composite kidney outcome after 1 year of randomization. Covariates used in multivariable Cox proportional analysis were in general weakly correlated with the change in uEGF / Cr, except for baseline uEGF / Cr (Figure 7). When adjusted for all covariates, each doubling of uEGF / Cr from baseline to year 1 significantly associated with a decreased risk of subsequent kidney outcomes with corresponding HR of 0.79 (95% CI 0.72, 0.85; p<0.01; Table 4). When uGFR / Cr was categorized in quartiles, the two upper quartiles where participants had an increase in uEGF / Cr exhibited a 2-fold lower risk of experiencing the composite kidney outcome compared to the second quartile with a modest decrease in mean uEGF / Cr from baseline (Table 4). TheThe change in uEGF / Cr at year 1 was independently associated with the composite kidney outcome in both the canagliflozin and placebo group (Table 6).

[0112] Exploration of molecular mechanism underlying uEGF / Cr association with improved kidney outcome after SGLT2i treatment

[0113] To understand the molecular mechanisms underlying the association of uEGF / Cr with improved kidney outcome in patients treated with the SGLT2 inhibitor, EGF mRNA levels following SGLT2 inhibition were investigated at the single cell kidney tissue level in protocol research biopsies in young persons with T2D at risk of diabetic kidney disease (DKD). Healthy young persons were also included. The baseline characteristics of participants were published previously (Schaub et al., JCI 2023).

[0114] Analysis of the scRNA-seq data from 22 participants resulted in 50,601cells that grouped into 18 cell clusters (Figure 3 A, Figure 8 A) representing the entire spectrum of kidney cell types along the nephron as well as tissue-resident immune cells, and each cell cluster had a robust representation of the 3 biopsy groups (Figure 8B).

[0115] The SLC5A2 gene, encoding the SGLT2 protein, was specifically expressed in the proximal tubular (PT) cell cluster (Figure 3C). EGF was abundantly expressed in the TAL and the DCT cell clusters with sporadic expression in the ascending thin loop of Henle (ATL) cell cluster (Figure 3B) cross three groups: healthy controls (HC) (n=6), 10 participants with T2D had been prescribed SGLT2i [T2Di(+)](n=10), and 6 not [T2Di(-)]. Consistent with previous reports (24-26), EGF is co-localized with the TAL-specific marker Uromodulin (UMOD) in TAL cells and with DCT-specific marker gene solute carrier family 12 member 3 (SLC12A3) in DCT cells (Figure 9).

[0116] To examine the influence of SGLT2i on intrarenal EGF expression and its downstream gene network, additional analyses were focused on the TAL cells representing the largest cell cluster that expresses EGF transcripts. As demonstrated in dot plot analyses, the mean expression of EGF mRNA in the TAL of participants not using SGLT2i, T2Di(-), was significantly lower compared to reference tissues from healthy participants HC (age adjusted p value = 1.94E-60, Table 7 and Figure 3C). In contrast, in T2Di(+) participants, we observed higher EGF mRNA expression level (age adjusted p value= 0.002, Figure 3D). The percentage of TAL cells expressing EGF (-90%) was not different between HCs and young persons with T2D using or not using SGLT2L

[0117] Impact ofSGLT2i on EGF co-expressing functional gene networks.

[0118] Differentially expressed transcripts between TAL cells with EGF expression (EGF+) (about 90% of the TAL cells) versus those without detectable EGF transcripts (EGF~) (approximately 10% of the TAL cells) were mapped to their cellular functional context (Figure 4). EGF positivity was defined as a normalized expression value of EGF greater than zero (EGF > 0).

[0119] In young persons with T2D using SGLT2 inhibitor treatment, 459 genes were identified that were differentially expressed between EGF+and EGF- TAL cells with an adjusted P value less than leA-20 and fold change above 1.20 or below 0.83. Of these, 402 genes (89.8%) had higher expression in EGF+TAL compared to the EGF TAL cells. UMOD, MALATl, XIST, CLDN10, CXCL12, DDX17, SLC12A1, NEAT1, KNG1 and HSP90B 1 were the top 10 genes among these highly expressed genes in EGF+TAL cells. In young persons with T2D not using SGLT2 inhibitors, only 183 genes were significantly differentially expressed between EGF+and EGF~ TAL cells, based on the same statistical and fold change cut offs.

[0120] To unravel the effect of SGLT2i on above functional gene networks in these 2 gene sets, we compared enriched molecular pathways between patients under standard care alone versus those treated with SGLT2i as well. We used IPA to reveal the enriched canonical pathways, gene networks, and upstream regulators (cytokines, growth factors, and transcriptional factors) in these 2 gene sets. The hierarchical graphic summary (Figure 4) demonstrates that endothelin-1 (EDN1) is the key regulator modulating gene and interacting networks derived from 183 genes differentially expressed between EGF+ and EGF- TAL cells in T2D patients under standard care only, whereas EGF was the factor placed on top of the hierarchical regulation cascade in T2D patients treated with a SGLT2i. We also performed a similar analysis on healthy control cells, but only 12 genes met the same cut-off criteria, making it difficult to perform a reliable enrichment analysis. To increase the number of genes for analysis, we relaxed the selection criteria to include genes with an adjusted P-value < 0.05 and fold change > 1.20 or < 0.83, resulting in the identification of 244 genes. Pathway analysis of these 244 genes showed that EGFR was the top node of the hierarchical regulation cascade in healthy individuals (Figure 10).

[0121] Table 1. Baseline characteristics of the total and placebo and canagliflozin treated population.

[0122] Total Placebo Canagliflozin

[0123] Characteristic (n = 3521) (n = 1181) (N = 2340)

[0124] Age, year 62.8 (7.8) 62.6 (7.8) 62.9 (7.8)

[0125] Male sex, n (%) 2352 (66.8) 790 (66.9) 1562 (66.8)

[0126] History of heart failure, n (%) 471 (13.4) 173 (14.7) 298 (12.7)

[0127] Duration of diabetes, year 13.6 (7.5) 13.3 (7.5) 13.7 (7.5)

[0128] History of CV disease, n (%) 2087 (59.3) 698 (59.1) 1389 (59.4)

[0129] BMI, kg / m232.7 (6.1) 32.6 (6.2) 32.7 (6.1)

[0130] Systolic BP, mmHg 136.7 (15.8) 137.2 (15.8) 136.4 (15.9)

[0131] Diastolic BP, mmHg 77.6 (9.8) 78.1 (9.8) 77.3 (9.7)

[0132] HbAlc mmol / mol 65.8 (10.0) 65.7 (9.9) 65.9 (10.0)

[0133] % 8.2 (0.9) 8.2 (0.9) 8.2 (0.9)

[0134] LDL, mmol / mL 2.3 (0.9) 2.3 (0.9) 2.3 (0.9)

[0135] HDL, mmol / mL 1.2 (0.3) 1.2 (0.3) 1.2 (0.3) eGFR, mL / min / 1.73 m277.0 (18.7) 76.9 (18.8) 77.0 (18.7) eGFR <60, n (%) 576 (16.4) 203 (17.2) 373 (15.9) eGFR >60, n (%) 2945 (83.6) 978 (82.8) 1967 (84.1)

[0136] UACR, mg / g, median (IQR) 11.6 (6.4, 35.1) 11.5 (6.3, 37.0) 11.6 (6.5, 34.4)

[0137] Normoalbuminuria, n (%) 2545 (72.3) 843 (71.4) 1702 (72.7)

[0138] Microalbuminuria, n (%) 776 (22.0) 257 (21.8) 519 (22.2)

[0139] Macroalbuminuria, n (%) 200 (5.7) 81 (6.9) 119 (5.1)

[0140] Continuous variables are reported as mean with SD unless noted as median IQR. Categorical variables are reported as quantity (n) with percentage.

[0141] Abbreviations: CV: cardiovascular; BMI: body mass index; BP: blood pressure; HbAlc: hemoglobin Ale; LDL: low-density lipoprotein; HDL: high-density lipoprotein; eGFR: estimated glomerular filtration rate; UACR: urine albumin-to-creatinine ratio; IQR: interquartile range; uEGF: urinary epidermal growth factor, uEGF / Cr: urinary epidermal growth factor to creatinine ratio; SD: standard deviation.

[0142] Table 2. Associations of baseline uEGF / Cr with the composite kidney outcome (40% eGFR decline / ESRD / renal death). Four models are presented adjusting for increasing number of covariates as presented in the figure legend. The table present analyses of the association between uEGF / Cr with the composite kidney outcome when uEGF / Cr is modeled as categorical (quartiles) and continuous (per doubling) variable.

[0143] Model 1 Model 2 Model 3 Model 4

[0144] HR (95% CI) P- value HR (95% CI) P- value HR (95% CI) P- value HR (95% CI) P- value uEGF / Cr

[0145] Quartile 1 (reference) (reference) (reference) (reference)

[0146] Quartile 2 0.7 (0.4, 1.0) 0.07 0.7 (0.5, 1.1) 0.10 0.7 (0.5, 1.1) 0.11 0.8 (0.6, 1.3) 0.46

[0147] Quartile 3 0.4 (0.2, 0.6) <0.01 0.4 (0.2, 0.6) <0.01 0.4 (0.2, 0.7) <0.01 0.5 (0.3, 0.8) 0.01

[0148] Quartile 4 0.3 (0.2, 0.6) <0.01 0.4 (0.2, 0.6) <0.01 0.4 (0.2, 0.6) <0.01 0.5 (0.3, 0.9) 0.03

[0149] Per doubling 0.8 (0.8, 0.9) <0.01 0.8 (0.8, 0.9) <0.01 0.9 (0.8, 0.9) <0.01 0.9 (0.8, 1.0) 0.04

[0150] Models are adjusted for the following covariates:

[0151] Model 1: Age, sex, race, and randomized treatment.

[0152] Model 2: Covariates of model 1 + HbAlc, systolic blood pressure, body mass index, LDL, HDL, and history of CV disease.

[0153] Model 3: Covariates of model 2 + baseline eGFR.

[0154] Model 4: Covariates of model 3 + log transformed baseline UACR.

[0155] Abbreviations: uEGF / Cr: urinary epidermal growth factor to creatinine ratio; HR: hazard ratio; CI: confidence interval; eGFR: estimated glomerular filtration rate; ESRD: end-stage renal disease; HbAlc: hemoglobin Ale; LDL: low-density lipoprotein; HDL: high-density lipoprotein; CV: cardiovascular; UACR: urine albumin-to-creatinine ratio.

[0156] Table 3. Effect of 52 weeks treatment with canagliflozin compared to placebo on uEGF / Cr. Data are presented in the overall population and in subgroups defined by baseline UACR, eGFR, MCP-1 and KIM-1.

[0157] Geometric mean baseline uEGF / Cr (ng / mg) Change from baseline at week 52 (%)

[0158] Canagliflozin Placebo Canagliflozin Placebo Between group P- value’ P for

[0159] (95% CI) (95% CI) difference (%) interactio

[0160] (95% CI)

[0161] Overall 5.10 5.25 -1.4 (-4.2, 1.4) -8.1 (-11.8, -4.2) 7.3 (2.0, 12.8)0.01

[0162] UACR 0.73

[0163] <30 5.45 5.45 -1.4 (-4.8, 2.1) -8.6 (-13.1, -3.9) 7.9 (1.5, 14.7) 0.02

[0164] >30 4.26 4.75 -1.5 (-6.1, 3.4) -6.3 (-12.8, -0.6) 5.2 (-3.5, 14.6) 0.25 eGFR 0.57

[0165] <60 3.21 3.96 -4.6 (-13.4, 5.1) -8.8 (-20.8, 5.1) 4.6 (-12.0, 24.2) 0.61

[0166] >60 5.52 5.52 -0.8 (-3.6, 2.0) -8.0 (-11.8, -4.1) 7.8 (2.5, 13.4) <0.01

[0167] MCP-1 0.85

[0168] <median 4.42 4.45 10.8 (6.1, 15.8) 4.0 (-2.5, 11.0) 6.6 (-1.4, 15.2) 0.11

[0169] >mcdian 5.91 6.15 -12.3 (-15.5, -9.0) -18.7 (-22.9, -14.4) 7.9 (1.2, 15.0) 0.02

[0170] KIM-1 0.39

[0171] <median 4.10 4.23 14.7 (9.6, 20.0) 4.6 (-2.1, 11.7) 9.7 (1.3, 18.8) 0.02

[0172] >median 6.30 6.48 -15.3 (-18.2, -12.3) -19.3 (-23.3, -15.2) 5.0 (-1.2, 11.6) 0.12

[0173] "P-value indicates the between group difference in uEGF.

[0174] Abbreviations: uEGF / Cr: urinary epidermal growth factor to creatinine ratio; CI: confidence interval; UACR: urine albumin-to-creatinine ratio; eGFR: estimated glomerular filtration rate; MCP-1: monocyte chemoattractant protein-1; KIM-1: kidney injury molecule-1; uEGF, urinary epidermal growth factor.

[0175] Table 4. Associations of the change in uEGF from baseline to year 1 with the composite kidney outcome (40% eGFR decline / ESRD / renal death). uEGF / Cr change from baseline was stratified in quartiles of change and analyzed as a categorical variable as well as a continuous variables with HRs expressed per doubling of uEGF from baseline to week 52.

[0176] Model 1 Model 2 Model 3 Model 4 Model s

[0177] Median HR (95% Cl) P-value HR (95% CI) P- value HR (95% CI) P-value HR (95% CI) -value HR (95% CI) -value change (%) uEGF / Cr

[0178] Quartile 1 -53.3 1.3 (0.9, 2.1) 0.21 1.2 (0.8, 1.9) 0.40 1.3 (0.8, 2.0) 0.26 1.3 (0.9, 2.1) 0.20 1.3 (0.8, 2.1) 0.22

[0179] Quartile 2 -12.9 reference reference reference reference reference

[0180] Quartile 3 8.4 0.3 (0.2, 0.6) <0.01 0.4 (0.2, 0.7) <0.01 0.4 (0.2, 0.7) <0.01 0.4 (0.2, 0.8) 0.01 0.4 (0.2, 0.8) 0.01

[0181] Quartile 4 41.5 0.3 (0.2, 0.6) <0.01 0.4 (0.2, 0.8) 0.01 0.4 (0.2, 0.7) <0.01 0.5 (0.2, 0.9) 0.02 0.4 (0.2, 0.8) 0.01

[0182] | Per doubling | | 0.8 (0.7, 0.9) | <0.01 | 0.8 (0.7, 0.9) | <0.01 | 0.8 (0.7, 0.9) | <0.01 | 0.8 (0.7, 0.9) | <0.01 | 0.8 (07, 0.9) | |

[0183] Models are adjusted for the following covariates:

[0184] Model 1: Baseline uEGF, age, sex, race and randomized treatment.

[0185] Model 2: Covariates of model 1 change in eGFR from baseline to year 1 and baseline eGFR.

[0186] Model 3: Covariates of model 1 change in UACRfrom baseline to year 1 and baseline UACR. Model 4: Covariates of model 1 change in eGFR and UACRfrom baseline to year 1 and baseline eGFR and UACR. Model 5: Covariates of model 1 history of CV disease, IlbAlc, systolic blood pressure, body mass index, LDL, IIDL, eGFR, baseline UACR and change in eGFR, UACR, systolic blood pressure, body mass index, and HbAl c from baseline to year 1. Abbreviations: uEGF: urinary epidermal growth factor; uEGF / Cr: uEGF to creatinine ratio; HR: hazard ratio; CI: confidence interval; eGFR: estimated glomerular filtration rate; ESRD: end-stage renal disease; CV: cardiovascular; HbAlc: hemoglobin Ale; LDL: low-density lipoprotein; HDL: high-density lipoprotein; UACR: urine albumin-to-creatinine ratio.

[0187] Table 5. Baseline characteristics of the total population by quartiles of uEGF.

[0188] Level of quartile

[0189] First Second Third Fourth

[0190] Characteristic Total (n = 3521)

[0191] (n=881) (n=88O) (n=880) (n=880)

[0192] Age, year 62.8 (7.8) 64.2 (7.7) 63.3 (7.6) 62.4 (7.7) 61.2 (7.9)

[0193] Male sex, n (%) 2352 (66.8) 690 (78.3) 634 (72.1) 568 (64.6) 460 (52.3)

[0194] History of heart failure, n (%) 471 (13.4) 108 (12.3) 104 (11.8) 112 (12.7) 147 (16.7)

[0195] Duration of diabetes, year 13.6 (7.5) 14.7 (7.7) 13.5 (7.5) 13.2 (7.5) 12.7 (7.3)

[0196] History of CV disease, n (%) 2087 (59.3) 539 (61.2) 544 (61.8) 521 (59.2) 483 (54.9)

[0197] BMI, kg / m232.7 (6.1) 32.8 (6.2) 32.3 (6.1) 32.6 (5.9) 33.0 (6.3)

[0198] Systolic BP, mmHg 136.7 (15.8) 138.2 (16.5) 136.2 (15.5) 135.7 (15.4) 136.5 (15.8)

[0199] Diastolic BP, mmHg 77.6 (9.8) 76.5 (10.0) 77.4 (9.6) 77.7 (9.6) 78.7 (9.6)

[0200] HbAlc mmol / mol 65.8 (10.0) 65.8 (9.9) 65.7 (9.8) 65.3 (9.7) 66.4 (10.5)

[0201] % 8.2 (0.9) 8.2 (0.9) 8.2 (0.9) 8.1 (0.9) 8.2 (1.0)

[0202] LDL, mmol / mL 2.3 (0.9) 2.1 (0.9) 2.3 (1.0) 2.3 (0.8) 2.4 (1.0)

[0203] HDL, mmol / mL 1.2 (0.3) 1.2 (0.3) 1.2 (0.3) 1.2 (0.3) 1.2 (0.3)

[0204] eGFR, mL / min / 1.73 m277.0(18.7) 67.3 (17.0) 75.2(17.4) 80.1 (17.1) 85.4(18.5) eGFR <60, n (%) 576(16.4) 286(32.5) 144(16.4) 87 (9.9) 59(6.7) eGFR>60,n(%) 2945 (83.6) 595 (67.5) 736(83.6) 793(90.1) 821 (93.3)

[0205] UACR, mg / g, median (IQR) 11.6(6.4,35.1) 16.6(7.1,67.2) 12.1 (6.7,35.5) 10.6(6.1,26.8) 9.6(6.1,23.1)

[0206] Normoalbuminuria, n (%) 2545 (72.3) 540(61.3) 637(72.4) 672(76.4) 696(79.1)

[0207] Microalbuminuria, n (%) 776(22.0) 255 (28.9) 185(21.0) 177(20.1) 159(18.1)

[0208] Macroalbuminuria, n (%) 200(5.7) 86(9.8) 58 (6.6) 31 (3.5) 25 (2.8) uEGF, pg / mL 4570 (2608, 7777) 2070 (1343, 3134) 4065 (2631, 5570) 5768 (3843, 8390) 9379 (6062, 14031) uEGF / Cr, ng / mg, median (IQR) 5.1 (3.4, 7.8) 2.4 (1.7, 2.9) 4.2 (3.8, 4.7) 6.3 (5.7, 7.1) 10.7(9.0, 14.2)

[0209] Continuous variables are reported as mean with SD unless noted as median IQR. Categorical variables are reported as quantity (n) with percentage.

[0210] Abbreviations: CV: cardiovascular; BMI: body mass index; BP: blood pressure; HbAlc: hemoglobin Ale; LDL: low-density lipoprotein; HDL: high- density lipoprotein; eGFR: estimated glomerular filtration rate; UACR: urine albumin-to-creatinine ratio; IQR: interquartile range; uEGF: urinary epidermal growth factor, uEGF / Cr: urinary epidermal growth factor to creatinine ratio; SD: standard deviation.

[0211] Table 6. Associations of the change in uEGF from baseline to year 1 with the composite kidney outcome (40% eGFR decline / ESRD / renal death) separate for the placebo and canagliflozin treated groups. uEGF / Cr change from baseline was stratified in quartiles of change and analyzed as a continuous variables and HRs expressed per doubling of uEGF from baseline to week 52.

[0212] Placebo (n=869;

[0213] Model 1 Model 2 Model 3 Model 4 Model 5 events=48)

[0214] Median HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value change (%) uEGF / Cr

[0215] Quartile 1 -75.8 1.2 (0.6, 2.4) 0.53 1.1 (0.5, 2.1) 0.82 1.4 (0.7, 2.8) 0.36 1.3 (0.6, 2.6) 0.49 1.2 (0.6, 2.5) 0.58

[0216] Quartile 2 -14.1 reference reference reference reference reference

[0217] Quartile 3 8.2 0.3 (0.1, 0.9) 0.03 0.3 (0.1, 0.9) 0.04 0.3 (0.1, 0.9) 0.03 0.3 (0.1, 0.9) 0.04 0.4 (0.1, 1.1) 0.08

[0218] Quartile 4 77.4 0.6 (0.2, 1.5) 0.27 0.7 (0.3, 1.7) 0.44 0.8 (0.3, 1.9) 0.57 0.8 (0.3, 2.0) 0.64 0.6 (0.2, 1.7) 0.35

[0219] Per doubling 0.8 (0.7, 0.9) <0.01 0.8 (0.6, 1.0) 0.02 0.8 (0.6, 0.9) <0.01 0.8 (0.6, 0.9) 0.02 0.8 (0.6, 1.0) 0.03

[0220] Canagliflozin (n=1838;

[0221] Model 1 Model 2 Model 3 Model 4 Model 5 events=62)

[0222] Median HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value change (%) uEGF / Cr

[0223] Quartile 1 -74.0 1.4 (0.8, 2.5) 0.24 1.3 (0.7,23) 0.37 1.3 (0.7,23) 0.40 1.4(0.8,23) 0.27 13(0.7,2.4) 0.42

[0224] Quartile 2 -13.0 reference reference reference reference reference

[0225] Quartile 3 8.8 03 (0.2,0.8) 0.01 0.4 (0.2, 1.0) 0.04 03 (0.2,0.8) 0.01 0.4 (0.2, 1.0) 0.05 0.4 (0.2, 0.9) 0.03

[0226] Quartile 4 60.1 0.2 (0.1, 0.4) <0.01 03(0.1,0.7) <0.01 0.2(0.1,03) <0.01 03(0.1,0.7) 0.01 0.2 (0.1, 0.6) <0.01

[0227] Per doubling 0.8 (0.7, 0.9) <0.01 0.8 (0.7, 0.9) 0.01 0.8 (0.7, 0.9) <0.01 0.8 (0.7, 0.9) <0.01 0.8 (0.7, 0.9) 0.01

[0228] Table 7. Comparison of EGF expression in TAL clusters between three biopsy groups. Welch t-test was used to compare the differences and Bonferroni -adjusted p value was presented.

[0229] EGF_Age Adjusted T2DvsHC T2DivsHC T2DivsT2D

[0230] Log2FC -0.34 -0.29 0.04

[0231] Adj.p value 1.94E-60 4.51E-48 0.002

[0232] Table 8. Canonical pathways enriched in EGF coregulated transcripts in EGF+ versus EGF- TAL cells of SGLT2i treated patients with DKD.

[0233] References

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[0263] All publications, patents, patent applications and accession numbers mentioned in the above specification are herein incorporated by reference in their entirety. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications and variations of the described compositions and methods of the disclosure will be apparent to those of ordinary skill in the art and are intended to be within the scope of the following claims.

Claims

CLAIMSWe claim:

1. A method of treating or preventing kidney disease, comprising: a) assaying a urine sample from a subject diagnosed with kidney disease for the level of urine epidermal growth factor (uEGF); and b) administering an SGLT2 inhibitor to a subject with a level of uEGF below a predetermined threshold level.

2. A method of treating or preventing kidney disease, comprising: a) administering an SGLT2 inhibitor to a subject with kidney disease; and b) assaying a urine sample from said subject for the level of uEGF, wherein said assaying is performed after said administering.

3. A method of recommending a treatment course of action in a subject with kidney disease or at risk of kidney disease, comprising: a) assaying a urine sample from a subject diagnosed with kidney disease for the level of uEGF; and b) recommending treatment with a SGLT2 inhibitor when said level of uEGF is below a predetermined threshold level.

4. The method of claim 4, further comprising the step of administering said treatment.

5. The method of any of the preceding claims, wherein said uEGF is measured as a uEGF to creatinine ratio (uEGF / Cr).

6. The method of any of the preceding claims, wherein said SGLT2 inhibitor is selected from the group consisting of canagliflozin, dapagliflozin, cmpagliflozin, and ertugliflozin.

7. The method of any of the preceding claims, wherein said administering is at regular intervals for a period of time.

8. The method of any of the preceding claims, further comprising repeating said assaying one or more times during and / or after said administering.

9. The method of claim 8, wherein said assaying is repeated at least once over a period of at least one year.

10. The method of any of the preceding claims, wherein said threshold level is a uEGF / Cr of 6 ng / mg or less.

11. The method of any of the preceding claims, wherein said subject has type II diabetes.

12. The method of any of the preceding claims, wherein said administering increases the level of EGF in the kidneys of said subject.

13. The method of any of the preceding claims, further comprising administering an additional treatment for kidney disease to said subject.

14. A method of providing a prognosis to a subject with kidney disease or at risk of kidney disease, comprising: a) assaying a urine sample from a subject diagnosed with kidney disease for the level of uEGF; andb) providing a prognosis of increased risk of severe disease when said level of uEGF is below a predetermined threshold level.

15. The use of an SGLT2 inhibitor to treat or prevent kidney disease in a subject with a level of uEGF below a predetermined threshold level.

16. The continued use of an SGLT2 inhibitor to treat or prevent kidney disease in a subject with a level change of uEGF above a predetermined threshold level.

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