Patent Application
20140303251
Acute kidney injury (AKI) is common, costly and independently associated with increased risk of death1. Patient outcomes are directly related to AKI severity, including even minor changes in serum creatinine2. More severe AKI is an independent risk factor for death with mortality rates generally exceeding 30% and often exceeding 50% when it occurs in the setting of trauma, surgery or multiple organ dysfunction3. After acute injury, tubular epithelial cells die through both necrotic and apoptotic mechanisms, and cells that survive the insult dedifferentiate and proliferate to reconstitute a functioning nephron4. Strategies to increase the fraction of epithelial cells that survive the initial insult should be therapeutic, by providing a larger mass of epithelial progenitors during the repair phase.
Acute kidney injury remains a major health problem with few therapeutic options. Accordingly, there is a considerable need for new therapies and therapeutic agents that are effective for the treatment of AKI.
As shown herein, Nur77 is rapidly and strongly induced in proximal tubule epithelia by acute kidney injury using in vivo and in vitro experimental models. In Nur77 knockout studies, Nur77 induction after acute kidney injury promotes epithelial apoptosis, and the absence of Nur77 reduces apoptosis, prevents pro-inflammatory cytokine induction and ameliorates histologic damage, leukocyte infiltration and renal function. In addition, as shown herein, 9-cis-retinoic acid reduces Nur77 induction and ameliorates acute kidney injury in wild type mice in vivo, and this protection is Nur77-dependent in vitro. Thus, the experiments provided herein support retinoid-based modulation of Nur77 induction as a novel strategy for the treatment of acute kidney injury. Such injuries can include hypoxia-induced injuries, ischemic reperfusion injuries, toxic or septic injuries.
Accordingly, the present invention provides, in one embodiment, a method of inhibiting apoptosis in kidney epithelial cells after acute injury by modulating/inhibiting Nur77 induction/expression or function/activity in the cells. In a particular embodiment, Nur77 induction/expression is inhibited by contacting the cells with retinoic acid, or a derivative thereof.
In another embodiment, the invention provides a method of preventing or treating an acute kidney injury in a subject in need thereof, comprising administering to the subject an effective amount of a retinoic acid or retinoic acid derivative. In a particular embodiment, the acute kidney injury is an ischemic renal injury.
In a further embodiment, the invention provides a method of preserving a kidney for transplant, comprising contacting the kidney with a solution that comprises a retinoic acid or a retinoic acid derivative, such as a cis- or trans-retinoic acid.
The present invention provides an important new therapeutic target and effective therapeutic agents for the treatment of acute kidney injury and related conditions.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
After acute kidney injury, epithelial cells die both through necrotic and apoptotic mechanisms and there is intense interest in understanding the signaling pathways that regulate these cell death pathways in order to design new therapeutic strategies5,6. While progress has been made in defining the execution phase of kidney apoptotic cell death, the upstream signaling pathways that initiate epithelial apoptosis after injury are poorly understood. Nur77 represents a strong candidate for such an apoptotic switch protein after injury. Hypoxia induces Nur77 expression via hypoxia-inducible factor-1a (HIF-1a)7, a transcription factor strongly induced by ischemic renal injury8, and Nur77 itself enhances HIF-1a transcriptional activity9. Originally identified as an immediate early gene transiently induced by serum or growth factors10), Nur77 is a potent proapoptotic inducer during thymocyte selection11 and in epithelial cancer cells exposed to antineoplastic agents, including lung12, prostate13 and colon14 through direct interaction with Bcl-2. These observations have led to development of Nur77 agonists as potential anticancer treatments.15
While Nur77 binds DNA and directly activates gene transcription; its function also depends on subcellular localization: in the nucleus Nur77 functions as an oncogenic survival factor whereas Nur77 translocation to mitochondria results in direct binding to Bcl-2 and conversion to an apoptotic phenotype16. Proapoptotic Nur77 nuclear export can be mediated by heterodimerization with retinoid X receptor (RXR), a process that is inhibited by retinoic acid ligand binding to RXR17. Retinoids also negatively regulate Nur77 by potently suppressing its transcriptional induction 18. The observation that retinoic acid can ameliorate toxin-induced AKT suggests the possibility that this effect might be mediated by inhibition Nur7719.
As described herein, it is demonstrated for the first time that Nur77 is rapidly and strongly induced in proximal tubule epithelia by acute kidney injury using in vivo and in vitro experimental models. In Nur77 knockout studies described herein, it is demonstrated that Nur77 induction after acute kidney injury promotes epithelial apoptosis, and the absence of Nur77 reduces apoptosis, prevents pro-inflammatory cytokine induction and ameliorates histologic damage, leukocyte infiltration and renal function. Importantly, it is shown that 9-cis-retinoic acid reduces Nur77 induction and ameliorates acute kidney injury in wild type mice in vivo, and this protection is Nur77-dependent in vitro. Retinoid-based modulation of Nur77 induction offers a novel strategy for the treatment of acute kidney injury.
Unless defined otherwise, all technical and scientific, terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods.
In one embodiment, the invention provides a method of inhibiting apoptosis in kidney epithelial cells after acute injury. Apoptosis may be inhibited by modulating (e.g., inhibiting) Nur77 induction/expression (e.g., Nur77 transcription, heterodimerization of Nur77 with retinoid X receptor) or function/activity (e.g., translocation from nucleus to cytoplasm) in the kidney cells. In a preferred embodiment, the kidney cells are proximal tubule epithelial cells.
The acute injury can be a hypoxia-induced injury. In such injuries, the hypoxia may be caused, for example, by an ischemia reperfusion injury (e.g., a renal ischemia reperfusion injury).
Nur77 induction/expression may be inhibited by contacting the cells with retinoic acid, or a derivative thereof. The retinoic acid (RA) may be all-trans-retinoic acid or a cis-retinoic acid (e.g., 7-cis-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid, 13-cis-retinoic acid). In a particular embodiment, Nur77 induction/expression is inhibited by contacting the cells with 9-cis-retinoic acid.
Derivatives of retinoic acid include, but are not limited to, amides and esters of retinoic acid, as well as retinamide/retinyl ester mimics. All-trans isomeric form of retinamides and retinyl esters are characterized by Structural Formula (1) shown below:
where X is —NR′— or —O—; and R1 is a substituted or unsubstituted aliphatic or aryl group, or a substituted or unsubstituted non-aromatic heterocyclic group; and R′ is hydrogen or a substituted or unsubstituted aliphatic or aryl group, or a substituted or unsubstituted non-aromatic heterocyclic group. It is noted that other isomers of the molecules characterized by Structural Formula (1), for example, 7-cis, 9-cis, 11-cis, 13-cis, or any combination thereof, can also be used in the invention. 9-cis and 13-cis isomers are exemplified in Structural Formulas (2) and (3), where X and R1 are as defined above:
Typically, R1 is a substituted or unsubstituted aryl or lower alkyl group. Preferably, when X is —NR′—, R1 is a substituted aryl group, such as 4-hydroxy phenyl, 3-hydroxy phenyl or 2-hydroxy phenyl. More preferably, when X is —NR′—, R1 is 4-hydroxy phenyl. Alternatively, when X is —O—, R1 is preferably a substituted or unsubstituted lower alkyl group, such as methyl, ethyl, propyl or benzyl.
As used herein, the term “aliphatic group” is non-aromatic, consists solely of carbon and hydrogen and may optionally contain one or more units of unsaturation, e.g., double and/or triple bonds. An aliphatic group may be straight chained, branched, or cyclic (i.e., “cycloaliphatic”). When straight-chained or branched, an aliphatic group typically contains between about 1 and about 24 carbon atoms, more typically between about 1 and about 12 carbon atoms. Aliphatic groups are preferably lower alkyl groups or lower alkylene groups, which include C1-24 (preferably C1-C12) straight chained or branched saturated hydrocarbons. An alkyl group is a saturated hydrocarbon in a molecule that is bonded to one other group in the molecule through a single covalent bond from one of its carbon atoms. Examples of lower alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl and tert-butyl. An alkylene group is a saturated hydrocarbon in a molecule that is bonded to two other groups in the molecule through single covalent bonds from two of its carbon atoms. Examples of lower alkylene groups include methylene, ethylene, propylene, iso-propylene (—CH(CH2)CH2-), butylene, sec-butylene (—CH(CH3)CH2CH2-), and tert-butylene (—C(CH3)2CH2-).
As used herein, the term“aryl group” may be used interchangeably with “aryl,” “aryl ring,” “aromatic group,” and “aromatic ring. Aromatic groups include carbocyclic aromatic groups and heteroaryl rings. Examples of carbocyclic aromatic groups include phenyl, 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthacyl. The term “heteroaryl”, “heteroaromatic”, “heteroaryl ring”, “heteroaryl group” and “heteroaromatic group” refer to heteroaromatic ring groups having five to fourteen members, including monocyclic heteroaromatic rings and polycyclic aromatic rings in which a monocyclic aromatic ring is fused to one or more other carbocyclic or heteroaromatic aromatic rings. Heteroaryl groups have one or more ring heteroatoms. Examples of heteroaryl groups include 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-pyrazolyl, 4-pyrazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-triazolyl, 5-triazolyl, tetrazolyl, 2-thienyl, 3-thienyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, isoquinolinyl, indolyl, isoindolyl, acridinyl, or benzisoxazolyl.
The term “non-aromatic heterocyclic group” refers to non-aromatic ring systems typically having five to fourteen members, preferably five to ten, in which one or more ring carbons, preferably one to four, are each replaced by a heteroatom such as N, O, or S. Examples of non-aromatic heterocyclic groups include 3-1H-benzimidazol-2-one, 3-tetrahydrofuranyl, 2-tetrahydropyranyl, 3-tetrahydropyranyl, 4-tetrahydropyranyl, [1,3]-dioxalanyl, [1,3]-dithiolanyl, [1,3]-dioxanyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, N-azetidinyl, 1-azetidinyl, 2-azetidinyl, N-oxazolidinyl, 2-oxazolidinyl, 4-oxazolidinyl, 5-oxazolidinyl, N-morpholinyl, 2-morpholinyl, 3-morpholinyl, N-thiomorpholinyl, 2-thiomorpholinyl, 3-thiomorpholinyl, N-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, N-piperazinyl, 2-piperazinyl, N-piperidinyl, 2-piperidinyl, 4-piperidinyl, N-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, diazolonyl, N-substituted diazolonyl, 1-pthalimidinyl, benzoxanyl, benzopyrrolidinyl, benzopiperidinyl, benzoxolanyl, benzothiolanyl, benzothianyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, azabicyclopentyl, azabicyclohexyl, azabicycloheptyl, azabicyclooctyl, azabicyclononyl, azabicyclodecyl, diazabicyclohexyl, diazabicycloheptyl, diazabicyclooctyl, diazabicyclononyl, and diazabicyclodecyl. Also included within the scope of the term “non-aromatic heterocyclic group”, as it is used herein, is a group in which a non-aromatic heteroatom-containing ring is fused to one or more aromatic or non-aromatic rings, such as in an indolinyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the non-aromatic heteroatom-containing ring.
Suitable substituents on an aliphatic group, such as an alkyl or alkenyl group, and aryl group are those which do not substantially interfere, with the inhibiting activity of the disclosed compounds. Examples of suitable substituents include —OH, halogens (—Br, —Cl, —I, —F), —ORa, —O—CORa, —CORa, —CN, —NCS, —NO2, —COOH, —SO3H, —NH2, —NHRa, —N(RaRb), —COORa, —CHO, —CONH2, —CONHRa, —CON(RaRb), —NHCORa, —NRbCORa, —NHCONH2, —NHCONRaH, —NHCON(RaRb), —NRbCONH2, —NRbCONRaH, —NR′CON(RaRb), —C(═NH)—NH2, —C(═NH)—NHRa, C(═NH)—N(RaRb), —C(═NRc)—NH2, —C(═NRc)—NHRa, —C(═NRc)—N(RaRb), —NH—C(═NH)—NH2, —NH—C(═NH)—NHRa, —NH—C(═NH)—N(RaRb), —NH—C(═NRc)—NH2, —NH—C(═NRc)—NHRa, —NH—C(═NRc)—N(RaRb), —NRdH—C(═NH)—NH2, —NRd—C(═NH)—N(RaRb), —NRd—C(═NRc)—NH2, —NRd—C(═NRc)—NHRa, —NRd—C(═NRc)—N(RaRb), —NHNH2, —NHNHRa, —NHRaRb, —SO2NH2, —SO2NHRa, —SO2NRaRb, —SH, —SRa, —S(O)Ra, and —S(O)2Ra. In addition, an aliphatic group, such as an alkyl, or alkenyl group, can be substituted with substituted or unsubstituted aryl group to form, for example, an aralkyl group such as benzyl. Similarly, an aryl group can be substituted with a substituted or unsubstituted alkyl or alkenyl group. Ra—Rd are each independently an alkyl group, aromatic group, non-aromatic heterocyclic group or —N(RaRb), taken together, form a substituted or unsubstituted non-aromatic heterocyclic group.
In another embodiment, the invention provides a method of preventing or treating an acute kidney injury in a subject in need thereof, comprising administering to the subject an effective amount of a retinoic acid or retinoic acid derivative. The term “acute kidney injury” or “AKI” as used herein refers to a condition that is characterized clinically by a rapid reduction in kidney function resulting in a failure to maintain fluid, electrolyte and/or acid-base homoeostasis. Acute kidney injury is commonly defined by an abrupt (e.g., within 48 h) increase in serum creatinine, resulting from an injury or insult that causes a functional or structural change in the kidney. The AKI may be caused by, for example, ischemia (e.g., ischemia reperfusion injury), sepsis and/or toxins (e.g., nephrotoxic insults such as toxic chemicals). In a particular embodiment, the AKI is an ischemic AKI. AKI includes acute renal failure (ARF), prerenal AKI (e.g., prerenal azotemia), intrinsic AKI (e.g., intrinsic renal azotemia) and postrenal AKI (e.g., postrenal azotemia). Thus, the acute kidney injury may be an ischemic injury, a toxicant-induced injury, a septic injury or a hypoxia-induced injury, or a combination thereof.
As used herein, the terms “treat,” “treating,” or “treatment,” mean to counteract an acute kidney injury (AKI) to the extent that the AKI is improved according to a clinically-acceptable standard. An improvement in an acute kidney injury can be determined according to one or more of the following clinical standards: 1) reduction in serum creatinine, 2) increase in kidney function, 3) increase in urine output, 4) decrease in blood urea nitrogen. Other markers such as the expression levels of Nur77 can be used. Clinical improvement in AKI can also be determined using one or more alternate biomarkers, such as, for example, urinary neutrophil gelatinase-associated lipocalin (NGAL), urinary interleukin 18, urinary kidney injury molecule 1 (KIM-1) and cystatin C.
“Prevent,” “preventing,” or “prevention,” as used herein, mean reducing the probability/likelihood or risk of an acute kidney injury in a subject, delaying the onset of a condition related to an acute kidney injury in the subject, lessening the severity of one or more symptoms of an acute kidney injury in the subject, or any combination thereof. In general, the subject of a preventative regimen most likely will be categorized as being “at-risk”, e.g., the risk for the subject developing an AKI is higher than the risk for an individual represented by the relevant baseline population.
The term “subject” refers to a mammal, including primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent or murine species. Examples of preferred subjects include, but are not limited to, humans, including humans who have, or are at risk for developing, an acute kidney injury. Examples of high-risk groups for the development of AKI include individuals having increased serum creatinine levels and reduced urine output. Further examples of high-risk groups include patients undergoing vascular surgeries and those with history of hypertension.
As used herein, an “effective amount” is an amount sufficient to inhibit (e.g., reduce, prevent) Nur77 induction/expression and/or function/activity in kidney cells of a subject. In a particular embodiment, the kidney cells are kidney epithelial cells, preferably proximal tubule epithelial cells.
A “therapeutically effective amount” is an amount sufficient to achieve the desired therapeutic or prophylactic effect under the conditions of administration, such as an amount sufficient to inhibit (e.g., reduce, prevent) an acute kidney injury. The effectiveness of a therapy can be determined by one of skill in the art using standard measures and routine methods.
The amount of the retinoic acid or derivative thereof to be administered to a subject (e.g., an effective amount, a therapeutically effective amount) can be determined by a clinician using the guidance provided herein and other methods known in the art and is dependent on several factors including, for example, the particular agent chosen, the subject's age, sensitivity, tolerance to drugs and overall well-being. For example, suitable dosages for retinoic acid or derivatives thereof can be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.01 mg/kg to about 1 mg/kg body weight per treatment. Determining the dosage for a particular agent, patient and condition is well within the abilities of one skilled in the art. Preferably, the dosage does not cause, or produces minimal, adverse side effects (e.g., immunogenic response, nausea, dizziness, gastric upset, hyperviscosity syndromes, congestive heart failure, stroke, pulmonary edema).
A therapeutically effective amount of a retinoic acid or derivative thereof can be administered alone, or in combination with one or more other therapies (e.g., vasodilation, volume replacement, blood purification and renal replacement therapy) and/or therapeutic agents useful for preventing or treating AKI, including, but not limited to, diuretics, antiobiotics, calcium and glucose/insulin. Emerging pharmacological agents for the treatment of AKI which may be used in combination with retinoic acid or retinoic acid derivatives are discussed in Jo et al., Clinical Journal of the American Society of Nephrology 2:356-365 (March, 2007), the contents of which are incorporated herein by reference, and include nonselective and selective caspase inhibitors, minocycline, guanosine, pifithrin-α, PARP inhibitors, sphingosine 1 phosphate analog, adenosine 2A agonist, α-MSH, IL-10, fibrate, PPAR-γ agonist, minocycline, activated protein C, iNOS inhibitor, insulin, activated protein C, ethyl pyruvate, recombinant erythropoietin, hepatocyte growth factor, carbon monoxide release compound and bilirubin, endothelin antagonist, fenoldopam and ANP.
The retinoic acid or derivative thereof can be administered before, after or concurrently with one or more additional therapies or therapeutic agents. In some embodiments, the retinoic acid or derivative thereof and additional therapy or therapeutic agent are co-administered simultaneously (e.g., concurrently) as either separate therapies/formulations or as a joint therapy/formulation. Alternatively, the therapies or agents can be administered sequentially, as separate compositions, within an appropriate time frame as determined by the skilled clinician (e.g., a time sufficient to allow an overlap of the pharmaceutical effects of the therapies). The retinoic acid or derivative thereof and one or more additional therapies or therapeutic agents can be administered in a single dose or in multiple doses, in an order and on a schedule suitable to achieve a desired therapeutic effect. Suitable dosages and regimens of administration can be determined by a clinician and are dependent on the agent(s) chosen, pharmaceutical formulation and route of administration, various patient factors and other considerations.
A retinoic acid or derivative thereof can be administered to a mammalian subject in a pharmaceutical or physiological composition, for example, as part of a pharmaceutical composition comprising a retinoic acid or derivative thereof and a pharmaceutically acceptable carrier. Formulations or compositions (e.g., solution, emulsion or capsule) comprising a retinoic acid or derivative thereof or compositions comprising a retinoic acid or derivative thereof and one or more other therapeutic agents (e.g.,) will vary according to the route of administration selected.
Suitable pharmaceutical carriers can contain inert ingredients which do not interact with the retinoic acid or derivative thereof. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's lactate and the like. Formulations can also include small amounts of substances that enhance the effectiveness of the active ingredient (e.g., emulsifying, solubilizing, pH buffering, wetting agents). Methods of encapsulation compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art. For inhalation, the agent can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer or nebulizer or pressurized aerosol dispenser).
A retinoic acid or derivative thereof may be administered to a subject by a variety of routes, preferably in the form of a pharmaceutical composition adapted to a desired route. The preferred route will depend, in part, on the condition being treated. The compounds and compositions may, for example, be administered intravascularly, intramuscularly, subcutaneously, intraperitoneally, orally or topically. It will be obvious to those skilled in the art that the following dosage forms may comprise as the active ingredient, either compounds or a corresponding pharmaceutically acceptable salt of a compound. Preferred routes of administration for the retinoic acid or derivatives thereof include oral and intravenous administration.
A retinoic acid or derivative thereof may be administered parenterally, via injection. Parenteral administration can include, for example, intraarticular, intramuscular, intravenous, intraventricular, intraarterial, intrathecal, subcutaneous, or intraperitoneal administration. Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions or suspensions may be prepared from sterile powders or granules having one or more of the carriers mentioned for use in the formulations for oral administration. The compounds may be dissolved in polyethylene glycol, propylene glycol, ethanol, corn oil, benzyl alcohol, sodium chloride, and/or various buffers (e.g., sodium bicarbonate, sodium hydroxide).
For oral administration, the pharmaceutical compositions comprising a retinoic acid or derivative thereof may be in the form of, for example, a tablet, capsule, suspension or liquid. The composition is preferably made in the form of a dosage unit containing a therapeutically effective amount of the active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, the tablets and capsules can contain, in addition to the active ingredient, conventional carriers such as binding agents, for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica, or talc; disintegrants, for example potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations generally in the form of aqueous or oily solutions, suspensions, emulsions, syrups or elixirs may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous agents, preservatives, coloring agents and flavoring agents. Examples of additives for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.
The invention further provides a method of preserving a kidney (e.g., a human kidney) for transplant (e.g., cold organ preservation), comprising contacting the kidney with a solution that comprises a retinoic acid or a retinoic acid derivative. Preferably, the retinoic acid or retinoic acid derivative is 9-cis-retinoic acid or 13-cis-retinoic acid. The solution comprising the retinoic acid or derivative thereof may include one or more additional components, such as, for example, dexamethasone, insulin, penicillin G, glutathione, allopurinol, or a combination thereof.
A description of example embodiments of the invention follows.
C57B6/J and Nur77 knockout mice were purchased from Jackson laboratories. Animals were housed in the AALAC-approved animal facility in a pathogen free environment. All the animal studies were approved by the institutional review board at the Beth Israel Deaconess Medical Center.
Male mice of age 8-10 weeks were used in this study. Animals were anesthetized using xylazine and ketamine and kept on 37° C. warming pads. The kidneys were exposed by flank incision and both kidney pedicles were clamped for 45 minutes using non-traumatic microaneurysm clamps (Roboz, Rockville, Md.). Reperfusion was confirmed visually and the incision was sutured. Body temperature of the animals was maintained at 37° C. throughout the procedure.
9-cis retinoic acid in DMSO was administered to animals at a dose of 10 mg/kg body weight, 4 h prior to the IRI procedure. The final concentration of DMSO was 25% (v/v). Control animals received 25% (v/v) DMSO.
Kidneys from animals subjected to IRI were harvested. Transverse sections of the kidneys were either snap frozen in OCT or fixed in 10% formalin and processed for paraffin embedded sections. Renal morphology was assessed by PAS staining (Periodic acid-Schiff reagent). Neutrophil infiltration was assessed by staining formalin fixed paraffin embedded (FFPE) sections with anti-Ly6G (BD). Infiltrating neutrophils were counted in 3 independent HPFs (400×) per section per animal and represented as mean±SEM. Nur77 staining was carried out on FFPE sections after antigen retrievel using anti-mouse Nur77 antibody (Ebioscience). Endogenous mouse Ig was blocked using M.O.M kit from vector labs.
Kidney cell apoptosis was assessed by TUNEL staining using ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Millipore). The apoptotic index was calculated as percentage of dead cells in 3 independent 400×HPF per section per animal by, counting both stained nuclei and total nuclei.
Serum plasma was collected 24 h after ischemia reperfusion and stored at −80 deg C. until further processing. Serum creatinine was quantified using QuantiChrom™ Creatinine Assay Kit (Bioassays systems). Serum blood urea nitrogen levels were measured using Infinity Urea liquid stable reagent (Thermo fisher).
Kidney tissues were snap frozen and stored at −80 deg C. until processing. The tissues were homogenized and total RNA was prepared using RNeasy kit (Qiagen). DNA contamination was eliminated by DNase digestion. The reverse transcribed cDNA was subjected to qPCR using a Sybr green based detection system (SA Bioscience). Relative levels of mRNA was normalized to GAPDH levels and quantified based on 2e-deltaCT method.
Human Kidney Proximal tubular epithelial cell line, HK2 was purchased from ATCC and maintained in Keratinocyte-Serum Free Medium (K-SFM) at 37 deg C. with 5% CO2. To perform chemical induced IRI, cells were washed with Hank's Buffered Salt Solution (HBSS) and incubated with HBSS containing Antimycin (10 uM), 2-Deoxy Glucose (10 mM) and A23187 (2 uM) for 1 hour at 37 deg C., 5% CO2. Subsequently the cells were washed with HBSS and cultured in complete culture medium for defined period of time.
Nur77 RNA expression was localized by RNA in situ hybridization following the protocol as described elsewhere33. Nur77 riboprobe corresponds to 300 by nucleotides complementary to 581-880 by of the Nur77 coding sequence.
Kidneys from 4-5 weeks old mice were harvested after perfusion with sterile PBS. Cortex was separated and minced into ˜1 mm3 pieces and digested using collagenase and soybean trypsin inhibitor for 30 min and 37 degC. The digested mix was spun down and filtered through 200 uM filter followed by a 70 uM filter. The tubules that are retained on the top of the 70 uM filter were collected and cultured in Renal life cell growth medium (Lifeline cell technology) on collagen coated plates. After 7 days of culturing, the cells were used for experiments. The purity of the culture was ascertained by staining for cytokeratin (Anti-cytokeratin antibody: Sigma). To induce ischemia reperfusion injury, confluent monolayer of RPTECs was overlayed with mineral oil for 1 h, followed by reperfusion with complete medium.
All comparisons were performed using a two-tailed unpaired Student's t test.
The observation that NR4a members are induced at the mRNA level in whole kidney lysates either by parathyroid hormone injection20 or in a glomerular disease model21 prompted us to examine a possible role for NR4a family members in acute kidney injury. Using the murine IRI model of acute kidney injury, the expression of Nur77, Nurr1 and Nor-1 was examined during the early phases of AKI. Nur77 was very rapidly and strongly induced after renal IRI, with a lower induction of both Nor-1 and Nurr1 (
The renal cell type expressing Nur77 after acute injury was explored. To assess whether Nur77 is capable of being induced in proximal tubule, the major tubule segment present in outer medulla, primary proximal tubule kidney cultures (
To define the identity of cells that express Nur77 mRNA after acute kidney injury in vivo, kidneys from wildtype or Nur77 null mice were subject to in situ hybridization for Nur77 message after renal injury. As expected, Nur77 was undetectable in sham-operated kidneys. Three hours after renal IRI, Nur77 signal was present in cortical tubules that showed evidence of damage, in medullary tubules and in papilla (
To examine the functional role of Nur77 in renal IRI, the effect of renal IRI between wild type and Nur77 knockout mice which have no detectable phenotype at baseline was compared 22. 24 hours after injury, wild type mice had substantially worse renal function than Nur77− littermate controls as reflected by higher serum BUN and creatinine values (
Since epithelial apoptosis is an important consequence of ischemic injury and Nur77 can be proapoptotic in many cellular contexts, tubular apoptosis was next assessed by TUNEL staining. There was a significant reduction of TUNEL-positive tubular nuclei in Nur77− kidneys compared to controls by 50% (
Reduced Proinflammatory Mediators in Nur77 Deficient Mice after Acute Kidney Injury
Epithelial apoptosis after renal IRI is a pro-inflammatory stimulus through elaboration of soluble mediators that activate and recruit leukocytes23. Since previous results indicated that Nur77 promotes epithelial apoptosis and neutrophil accumulation, it was hypothesized that Nur77-deficient mice that are characterized by reduced epithelial apoptosis would also elaborate fewer proinflammatory cytokines. Levels of these cytokines were assessed by qPCR. mRNA encoding CCL20, CXCL10, CSF-1, IL-6 and TGF β, all cytokines capable of being secreted by damaged epithelia were substantially inhibited in Nur77 deficient kidney lysates (
These results, however, do not distinguish between Nur77-dependent promotion of apoptosis and Nur77-mediated stimulation of inflammation. To distinguish between these possibilities we examined the expression pattern of Cxcl2, a chemokine that is significantly diminished in the Nur77 null mice (
Nur77 dependent apoptotic induction in cells can convert pro-survival Bcl2 to a proapoptotic molecule by exposing its BH3 domain via conformational change. Immunostaining of kidney tissues with a specific anti-BH3 domain antibody demonstrated pronounced exposure of the BH3 domain of Bcl2 in wild type, but not Nur77 KO or sham operated kidneys (
Retinoic acid can antagonize Nur77 function by inhibiting its transcriptional induction, DNA binding and nuclear export17,18. RXRs heterodimerize with Nur77 in response to cell death stimuli, promoting nuclear export and causing Bcl-2 dependent cytochrome c release and apoptosis. Ligand bound RXR inhibits Nur77 function either by retaining it in the nucleus or by inhibiting its transcription by an AP-1 dependent mechanism18. Since Nur77 deficient mice were protected from renal IRI, whether pharmacologic antagonism of Nur77 by retinoic acid could be used to protect kidneys from acute injury was assessed. To determine whether retinoids could regulate Nur77-dependent signaling in renal epithelia, primary cultures of renal proximal tubule epithelial cells (RPTECs) were generated from either wild type or Nur77 knockout mice. RPTECs were treated with either vehicle or 9-cis-retinoic acid, and subjected to mineral oil overlay induced hypoxia, followed by reperfusion. Nur77 levels were reduced by about half in 9-cis-retinoic acid-treated cultures (
Nur77 Antagonism with Retinoic Acid Receptor Ligand Ameliorates Acute Kidney Injury
To investigate whether 9-cis-retinoic acid would protect from renal IRI, mice were treated with vehicle 9-cis retinoic acid, four hours prior to renal IRI. 24 hours after injury, mice that received 9-cis retinoic acid had dramatically lower induction of all three NR4a members Nur77, Nor-1 and Nurr-1 (
Since retinoids are capable of inhibiting Nur77 by several mechanisms including both inhibition of transcriptional induction and preventing nuclear to cytoplasmic translocation, the Nur77 protein expression and subcellular localization in injured kidney was investigated. While Nur77 was undetectable in uninjured kidneys, one hour after renal IRI Nur7 could be detected in a nuclear pattern in epithelial cells. By 24 hours after injury, Nur77 expression was cytoplasmic, consistent with its known proapoptotic effects in cytoplasm (
Acute kidney injury remains a major health problem with few therapeutic options. The present study provides the first demonstration that Nur77 is rapidly and strongly induced in injured proximal tubule epithelia where it regulates apoptosis. Epithelial apoptosis after injury triggers a pro-inflammatory cascade resulting in tissue damage, and inhibition of apoptosis is a protective therapeutic strategy in preclinical and clinical settings23-25. These findings implicate Nur77 as a key regulator of both epithelial apoptosis but also of inflammation caused by injury-induced cell death. Furthermore, these results identify 9-cis retinoic acid as a protective therapy in acute kidney injury through inhibition of Nur77 induction, providing a novel therapeutic target for treatment of acute kidney injury.
Nur77 was induced in proximal tubule epithelial cells based on two lines of evidence. Primary cultures of proximal tubule epithelia were capable of inducing Nur77 expression after hypoxia, and Nur77 mRNA was found in injured proximal tubules by in situ hybridization. These results are consistent with an earlier observation that systemic parathyroid hormone injection causes increased kidney expression of Nur7720, because proximal tubules express the parathyroid hormone receptor26. The induction of Nur77 in proximal tubules, the nephron segment most susceptible to hypoxic insult, suggests a role for Nur77 in mediating the stress response in injured epithelia.
The ability to pharmacologically antagonize Nur77 with retinoids represents an attractive therapeutic strategy. Retinoic acid has been shown to protect from toxicant-induced renal injury19, but the mechanism is unknown. These results suggest that in kidney, retinoids repress stress-induced Nur77 transcriptional induction. Retinoic acid is known to inhibit transcriptional induction of Nur77 by an AP-1-dependent mechanism in lymphocytes18, consistent with the observation of reduced expression of Nur77 mRNA and protein after retinoid treatment both in vitro and in vivo. In addition, Nur77 is capable of heterodimerization with ligand-bound retinoic acid receptor18, which prevents its nuclear export. Nuclear retention of Nur77 prevents its translocation to mitochondrial and cytochrome c release, preventing apoptosis13,17. RXR is a critical regulator of Nur77 translocation and apoptosis in response to a variety of extracellular ligands and stimuli (reviewed in 27), and the RXR nuclear export signal is conformationally regulated by retinoic acid ligand17. However, no change was seen in the pattern of nuclear to cytoplasmic Nur77 localization in kidney after 9-cis retinoic acid treatment, suggesting that retinoids act solely to repress Nur77 induction rather than to regulate its nuclear export. Combined with evidence that anti-apoptotic strategies can be therapeutic in other preclinical models of ischemia-reperfusion injury28-30, these results provide a basis for further exploration of the therapeutic potential of Nur77-targeted therapies in acute kidney injury.
The mechanism by which Nur77 expression in tubule epithelia triggers apoptosis requires further study, but may involve Bcl-2. While it typically functions as a survival factor, Bcl-2 is a mitochondrial receptor for Nur77. This interaction triggers a conformational change in Bcl-2, exposing its pro-apoptotic BH3 domain, causing cytochrome c release and apoptosis16. BH3-only proteins exert proapoptotic effects through either Bax or Bak31, and indeed Nur77-dependent apoptosis is dependent on Bak in lung cancer cells16. Bax is proapoptotic in renal epithelial cells, where it is regulated by cell survival kinases Akt and glycogen synthase kinase 3β (GSK3β)32. The finding that Nur77 expression promotes epithelial apoptosis, and that Nur77 translocates from nucleus to cytoplasm during renal injury is consistent with a model involving Bcl-2-dependent proapoptotic signaling. Whether Nur77-dependent apoptosis in renal epithelial cells depends on multidomain proapoptotic Bcl-2-family proteins like Bax or Bak requires further investigation.
In summary, the data shown herein indicate a novel role for Nur77 in promoting epithelial apoptosis and inflammation after acute kidney injury. The ability to antagonize Nur77 function with retinoic acid ligands and thereby ameliorate ischemic renal injury represents a novel therapeutic strategy for this important clinical syndrome.
The relevant teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 61/437,971, filed on Jan. 31, 2011. The entire teachings of the above application are incorporated herein by reference.
This invention was made with government support under grants DK073628, DK088923 and P01 A73748 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/023233 | 1/31/2012 | WO | 00 | 6/5/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61437971 | Jan 2011 | US |
