Use of l-carnitine as an activator of the aim protein in felines and for improving kidney function in felines

Abstract

The application belongs to the technical field of animals, and particularly relates to the use of L-carnitine as an AIM protein activator for felines and the use for improving kidney function of felines. The application provides the use of L-carnitine or a physiologically acceptable salt thereof as an AIM protein activator for felines, the use for improving kidney function of felines, and the use in a composition for improving kidney function, preventing or treating kidney disease of felines; and the application also provides a composition for improving kidney function, preventing or treating kidney disease of felines, which comprises L-carnitine or a physiologically acceptable salt thereof. The application finds that L-carnitine can promote the dissociation of IgM-AIM complex in cat serum and activate AIM protein through research; therefore, L-carnitine can be used as an AIM protein activator for improving kidney function, preventing or treating kidney disease of felines, and has a wide application prospect.

Description

Technical Field

[0001] This application belongs to the field of animal technology, specifically relating to the use of L-carnitine as an AIM protein activator in felines and its use in improving renal function in felines. Background Technology

[0002] AIM (apoptosis inhibitor of macrophage, encoded by the cd5l gene) is a circulating protein in the blood. AIM circulates in an inactive state by binding to the pentamer of immunoglobulin M (IgM) in the blood. During acute renal failure, AIM dissociates from IgM and enters the kidney, marking dead kidney cells and triggering macrophages to recognize and phagocytose dead cells and other debris in order to alleviate and restore kidney inflammation.

[0003] Cats possess two types of AIM protein, 3SRCR and 4SRCR, with different molecular weights of approximately 37 and 49 kDa, respectively (Reference: Scientific Reports, volume 6, Article number: 35251 (2016)). Their amino acid sequences differ from those of humans by a key amino acid cluster. This charged amino acid cluster causes AIM to bind tightly to IgM, 1000 times tighter than in mice, making it difficult to dissociate. AIM cannot be carried into the urine and cannot enter the kidneys to perform its waste cell labeling function, resulting in the inability to break down and clear dead cells and other debris from the body. This means that from birth, all dead cells and debris produced in a cat's body accumulate in its kidneys, eventually leading to kidney strain and a high risk of acute kidney injury (AKI) or other kidney diseases. Therefore, the difficulty in activating AIM protein is the reason for the high incidence of feline kidney failure. If the feline AIM protein can be separated from IgM and the AIM protein can be activated, macrophages can effectively clear dead cells and debris in the body, thereby reducing the occurrence of kidney inflammation and nephropathy, and it is expected to prolong the lifespan of cats (References: Scientific Reports, volume 6, Article number: 35251 (2016); Seminars in Immunopathology (2018) 40: 567-575).

[0004] In 2016, Professor Miyazaki's team reported the following research findings: Researchers used "AIM-feline mice" as their research subjects. In these mice, the SRCR3 binding domain key cluster of the AIM protein was expressed in a cat-like structure, exhibiting similar behavior to AIM in cats, and both were difficult to dissociate from IgM. These mice were then induced to develop symptoms of acute kidney injury and treated with recombinant AIM to observe the effects. The results showed that when ischemia / reperfusion (IR) induced acute kidney failure (AKI) occurred, AIM-deficient (AIM-) mice were unable to clear debris, exhibiting persistent kidney inflammation, and their mortality rate due to progressive renal dysfunction was higher than that of wild-type mice. Treatment of IR-induced AKI mice with recombinant AIM could clear debris, improve kidney pathology, and increase survival rate. Therefore, injecting recombinant AIM into mice with acute kidney injury has a certain therapeutic effect and improves the survival rate of mice (Reference: Scientific Reports, volume 6, Article number: 35251 (2016)).

[0005] A feline drug that involves direct injection of recombinant AIM protein is currently undergoing clinical trials in Japan. In a 23-year-old cat with kidney disease, four months of continuous injections of AIM helped clear blood waste and prevent the kidney disease from worsening, but it could not restore the cat's kidney function.

[0006] In the field of feline epidemiology, a research paper on the causes of feline mortality reported the following: 3108 cats that underwent autopsies at the William R. Pritchard Veterinary Teaching Hospital at the University of California, Davis, between January 1, 1989, and October 31, 2019, were used as subjects to determine lifespan and factors influencing mortality. This feline epidemiology study assessed sex, environmental factors, age, and cause of death. The sex distribution included females (5.66%), spayed females (39.86%), males (6.95%), and spayed males (47.49%), with 84.2% being mixed breeds. The age of 2974 cats was determined, with a median of 9.07 years. The results showed that cancer was the most common cause of death (35.81%), and 62.84% of the cats had kidney abnormalities (but only in 13.06% of the cats did kidney abnormalities become the primary cause of death). This authoritative study indicates that 62.84% of the cats had kidney problems, meaning that over 60% of the cats autopsied had chronic kidney disease. This corroborates Professor Miyazaki's discovery of defects in the AIM protein in felines.

[0007] Therefore, cat food and health products based on the AIM mechanism, such as prescription renal food, as well as drugs for the prevention or treatment of kidney disease, have a solid scientific research foundation and broad market demand.

[0008] Currently, only a series of food products targeting feline AIM protein are available in Japan, mainly including the following:

[0009] AIM30 series cat food: Developed by comprehensive pet food manufacturer Markan, it contains the "A-30" amino acid ingredient that can activate AIM proteins in cats. These products are designed to prevent kidney disease, especially for kittens that have not yet developed kidney damage.

[0010] AIM Nutritional Supplement: The product name is "Japanese Domestic AIM 30 Cat Kidney Health Nutritional Supplement". It is a powder supplement that allows cats to take the amino acid "A-30", which can effectively activate AIM in the cat's body and promote the elimination of waste products.

[0011] Cat treats: In addition to cat food and nutritional supplements, there are also cat treats with added AIM protein-related ingredients, which are also designed to support the kidney health of cats.

[0012] The existing AIM30 series of foods contains "A-30" amino acids, which, according to food formulation analysis, include a combination of cystine, methionine, taurine, etc. Methionine and taurine are both essential amino acids added to cat food. Whether these amino acids can affect the AIM protein activation function in cats has not yet been reported. Finding more effective AIM protein activators that can be used in cat food or health products is of positive significance for improving the high incidence of kidney disease and abnormal kidney function in cats. Summary of the Invention

[0013] Addressing the technical challenges of high incidence and functional abnormalities in feline kidney disease, this application has discovered through research that L-carnitine can promote the dissociation of the IgM-AIM complex in feline serum and activate AIM protein. Therefore, L-carnitine, as an AIM protein activator, can be used to improve renal function in felines or to prepare compositions that improve renal function, prevent or treat kidney disease, such as prescription diets (e.g., kidney prescription diets), foods (staple foods, snacks, etc.), food additives, health products, nutritional supplements, etc., to improve renal function, repair kidney damage, and prevent or treat kidney disease.

[0014] In a first aspect, this application provides the use of L-carnitine or a physiologically acceptable salt thereof as an activator of feline AIM protein or in the preparation of a feline AIM protein activator.

[0015] In some embodiments, the AIM protein activator can promote the dissociation of the IgM-AIM complex.

[0016] In a second aspect, this application provides the use of L-carnitine or a physiologically acceptable salt thereof for improving renal function, preventing or treating kidney disease in felines. In some embodiments, the L-carnitine or a physiologically acceptable salt thereof may be administered to felines via the gastrointestinal tract. The gastrointestinal administration to felines may be via oral administration.

[0017] In a third aspect, this application provides the use of L-carnitine or a physiologically acceptable salt thereof in compositions for improving renal function, preventing or treating kidney disease in felines; or the use of L-carnitine or a physiologically acceptable salt thereof in the preparation of compositions for improving renal function, preventing or treating kidney disease in felines.

[0018] In some embodiments, the composition can be administered to felines via the gastrointestinal tract or parenteral route. Gastrointestinal administration to felines can be via oral administration, while parenteral administration can be via intravenous, subcutaneous, arterial, or intraperitoneal administration.

[0019] In some embodiments, the composition is a prescription food, food product, food additive, health product, or nutritional supplement. The food product includes staple food, snacks (such as canned food, cat treats, freeze-dried food, etc.).

[0020] In a fourth aspect, this application provides a composition for improving renal function, preventing or treating kidney disease in felines, comprising L-carnitine or a physiologically acceptable salt thereof.

[0021] In some embodiments, a composition for improving kidney function, preventing or treating kidney disease in felines further comprises one or more of L-cysteine ​​or a physiologically acceptable salt thereof, and / or vitamin B1 or a physiologically acceptable salt thereof.

[0022] In some embodiments, a composition for improving kidney function, preventing or treating kidney disease in felines comprises L-carnitine or a physiologically acceptable salt thereof, and vitamin B1 or a physiologically acceptable salt thereof; or comprises L-cysteine ​​or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof; or comprises L-cysteine ​​or a physiologically acceptable salt thereof, vitamin B1 or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof.

[0023] In some embodiments, a composition for improving renal function and preventing or treating kidney disease in felines comprises L-carnitine or a physiologically acceptable salt thereof, and vitamin B1 or a physiologically acceptable salt thereof; wherein the weight ratio of L-carnitine or a physiologically acceptable salt thereof to vitamin B1 or a physiologically acceptable salt thereof is 0.0001-10000:1, 0.01-1000:1, 0.1-1000:1, 1-1000:1, 10-1000:1, preferably 20-500:1, more preferably 50-200:1, such as 100:1, 150:1, 200:1.

[0024] In some embodiments, a composition for improving renal function and preventing or treating kidney disease in felines comprises L-cysteine ​​or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof; wherein the weight ratio of L-cysteine ​​or a physiologically acceptable salt thereof to L-carnitine or a physiologically acceptable salt thereof is 1:0.0001-10000, 1:0.001-1000, 1:0.01-100, preferably 1:0.1-10, more preferably 1:0.2-5, such as 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5.

[0025] In some embodiments, a composition for improving renal function and preventing or treating kidney disease in felines comprises L-cysteine ​​or a physiologically acceptable salt thereof, vitamin B1 or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof; wherein the weight ratio of L-cysteine ​​or a physiologically acceptable salt thereof, vitamin B1 or a physiologically acceptable salt thereof, and L-carnitine or a physiologically acceptable salt thereof is (0.0001-10000): 1:(0.0001-1 0000), (0.01-1000):1:(0.01-1000), (0.1-1000):1:(0.1-1000), (1-1000):1:(1-1000), (10-1000):1:(10-1000), preferably (20-500):1:(20-500), more preferably (50-200):1:(50-200), such as 100:1:100, 150:1:150, 200:1:200.

[0026] In some embodiments, the composition can be administered to felines via the gastrointestinal tract or parenteral route. Gastrointestinal administration to felines can be via oral administration, while parenteral administration can be via intravenous, subcutaneous, arterial, or intraperitoneal administration.

[0027] In some embodiments, the composition is a prescription diet, food, food additive, health product, or nutritional supplement. The food includes staple food, snacks (such as canned food, cat treats, freeze-dried food, etc.). In some embodiments, the composition for improving kidney function and preventing or treating kidney disease in felines may also contain other ingredients used in prescription diets, food products, food additives, health products, or nutritional supplements.

[0028] In a fifth aspect, this application provides a method for improving renal function in felines, preventing or treating kidney disease by administering L-carnitine or a pharmaceutically acceptable salt thereof, or the composition described in the fourth aspect, to the feline.

[0029] In some embodiments, the L-carnitine or a pharmaceutically acceptable salt thereof, or the composition described in the fourth aspect, may be administered to felines via the gastrointestinal tract (e.g., orally) or parenterally (e.g., intravenously, subcutaneously, arterially, intraperitoneally, etc.).

[0030] In some embodiments, the method for improving renal function in felines, preventing or treating kidney disease, may administer L-carnitine or a pharmaceutically acceptable salt thereof, or the composition described in the fourth aspect, once or multiple times as appropriate. The dosage may be adjusted based on the feline being tested and its renal function.

[0031] In some embodiments, the method for improving renal function in felines, preventing or treating kidney disease, involves administering L-carnitine or a pharmaceutically acceptable salt thereof to the feline, or the composition described in the fourth aspect, wherein the composition is a prescription diet, food, food additive, health product, or nutritional supplement.

[0032] In some embodiments, the method for improving renal function in felines and preventing or treating kidney disease may involve a single administration of L-carnitine or a physiologically acceptable salt thereof at a dose of 0.01-1000, 0.1-1000, 1-1000, or 10-1000 mg / kg, preferably 20-500 or 50-200 mg / kg, such as 20, 30, 40, 50, 60, 80, 100, 150, or 200 mg / kg. Similarly, the single administration of L-cysteine ​​or a pharmaceutically acceptable salt thereof may be 0.01-10000, 0.1-1000, 1-1000, or 10-1000 mg / kg, preferably 20-500 or 50-200 mg / kg, such as 20, 30, 40, 50, 60, 80, 100, 150, or 200 mg / kg. The single dose of the vitamin B1 or its physiologically acceptable salt can be 0.01-1000, 0.1-100 mg / kg, preferably 0.2-50, 0.5-30 mg / kg, such as 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0 mg / kg.

[0033] The single dose mentioned in this application refers to a single dose based on the body weight of the feline, such as "0.01-1000mg / kg", which means a single dose of 0.01-1000mg per kg of feline body weight.

[0034] The feline animals mentioned in this application refer to animals classified in the Felidae family, such as cats, lions, tigers, leopards, etc., preferably cats, such as pet cats.

[0035] The improvement of renal function described in this application preferably refers to improving renal dysfunction, renal abnormality, renal insufficiency, and renal damage caused by the inability of the IgM-AIM complex to dissociate and the inability of AIM protein to be activated.

[0036] The kidney diseases described in this application include acute renal failure, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, IgM nephropathy, or kidney diseases related to collagen disorders. In some embodiments, the kidney disease is acute renal failure or chronic renal failure.

[0037] Compared with the prior art, the beneficial effects of this application are as follows:

[0038] This application, through in vitro and in vivo experiments, clearly verifies that L-carnitine can promote the dissociation of the IgM-AIM complex in feline serum and activate AIM protein. Free AIM protein can label dead cells and other debris, enabling macrophages to effectively clear dead cells and debris from the body, thereby improving kidney function, reducing kidney damage, and decreasing the incidence of kidney inflammation and nephropathy. Therefore, L-carnitine, as an AIM protein activator, can be used to improve kidney function and prevent or treat kidney disease in felines. It can be used to prepare compositions for improving kidney function and preventing or treating kidney disease in felines, such as prescription diets (e.g., renal prescription diets), foods, food additives, health products, and nutritional supplements, showing broad application prospects. Attached Figure Description

[0039] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein:

[0040] Figure 1 In vitro effect of L-cysteine ​​and methionine promoting the dissociation of the IgM-AIM complex.

[0041] Figure 2 In vitro effect of L-cysteine ​​promoting the dissociation of the IgM-AIM complex.

[0042] Figure 3A In vitro effects of different concentrations of L-cysteine ​​and cystine on the dissociation of the IgM-AIM complex.

[0043] Figure 3B In vitro effects of different concentrations of L-carnitine and cystine on promoting the dissociation of the IgM-AIM complex.

[0044] Figure 3C In vitro effects of different concentrations of vitamin B1 and cystine on promoting the dissociation of the IgM-AIM complex.

[0045] Figure 4A In vivo effect of L-cysteine ​​promoting the dissociation of the IgM-AIM complex.

[0046] Figure 4B This is an in vivo image illustrating the effect of L-carnitine on promoting the dissociation of the IgM-AIM complex.

[0047] Figure 4C An in vivo diagram illustrating the effect of vitamin B1 in promoting the dissociation of the IgM-AIM complex.

[0048] Figure 5 The in vivo effect of using L-cysteine, vitamin B1 and L-carnitine in combination to promote the dissociation of the IgM-AIM complex. Detailed Implementation

[0049] The specific embodiments of this application are described in detail below. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this application.

[0050] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0051] Features described or illustrated as part of one or more embodiments may be used in another or more embodiments to produce further embodiments.

[0052] Before describing this application in detail, it should be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of this application, which is defined solely by the appended claims. For a more complete understanding of the application described herein, the following terms are used, and their definitions are as follows. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this application pertains.

[0053] definition

[0054] Unless otherwise specified, the following terms used in this application shall have the following definitions.

[0055] Unless otherwise specified, the terms “comprising,” “including,” and “containing,” as well as similar expressions, shall be interpreted in an open and inclusive sense as “including but not limited to.”

[0056] The term “optional” means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event or situation occurs and the scenario in which it does not occur.

[0057] The term "AIM protein" refers to AIM protein (apoptosis inhibitor of macrophage, encoded by the cd5l gene), a circulating protein in the blood. AIM circulates in an inactive state by binding to immunoglobulin M (IgM) pentamers in the blood. During acute renal failure, AIM separates from IgM and enters the kidney, marking dead kidney cells and triggering macrophages to recognize and phagocytose dead cells and other debris in order to alleviate and restore kidney inflammation.

[0058] The feline AIM protein used in the embodiments of this application is a recombinant protein, approximately 37 kDa, and its amino acid sequence is as follows.

[0059] Seq ID No.1:

[0060] MGWSCIILFLVATATGVHSSFSRVRLVGGDHRCEGRVELQQDDEWVTVCDDYWNMDSVAVLCRELGCGAARKTMSGTVYGPVTPKDQKVFIHLFRCNGIEESLSQCEREDAIGCSHVEDAGAVCEPIYTGPGILGPESVRLADGPGRCQGRVEVKFRGEWSSVCQAGWSFAAAKVVCRQLG CGRATLTRRGCNKATQGQGAIWQRKASCSGQEVSLQDCLSEVWEHNCTHNEDVWVECEDPFALKLVGGRSHCEGRLEVLHKGEWGSVCDDGWGQDADRVVCRQLGCGQPLSPPVKVRRRFGPGVGRIWLDDVKCSGKEPSLEQCLHRSWGYHNCNHREDVAVVCEEQQSGLPDAHHHHHH*.

[0061] In the context of this invention, the term "physiologically acceptable salt" refers to any salt that is physiologically tolerable (most often meaning non-toxic – particularly lacking toxicity caused by counterions). When used in pharmaceuticals, it generally refers to a pharmaceutically acceptable salt. These physiologically acceptable salts can form with cations or bases, or with anions or acids.

[0062] The feline animals mentioned in this application refer to animals classified in the Felidae family, such as cats, lions, tigers, leopards, etc. For example: lion (Panthera leo), leopard (Panthera pardus), tiger (Panthera tigris), snow leopard (Panthera uncia), jaguar (Panthera onca), clouded leopard (Neofelis nebulosa), Bornean clouded leopard (Neofelis diardi), puma concolor, cheetah (Acinonyx jubatus), slender cat (Herpailurus yagouaroundi), African golden cat (Caracal aurata), Persian wildcat (Caracalcaracal), serval (Leptailurus serval), Bornean golden cat (Catopuma badia), golden cat (Catopumatemminckii), clouded leopard (Pardofelis marmorata), South American savanna cat (Leopardus colocola), George's cat (Leopardus geoffroyi), South American forest cat (Leopardus guigna), tabby cat (Leopardus guttulus), mountain cat (Leopardus Jaguar cat (Leopardus pardalis), jaguar cat (Leopardus tigrinus), long-tailed ocelot (Leopardus wiedii), Canadian lynx (Lynx canadensis), lynx (Lynx lynx), Iberian lynx (Lynx pardinus), hooked-tailed cat (Lynx rufus), leopard cat (Prionailurus bengalensis), Bornean leopard cat (Prionailurus javanensis), flat-headed leopard cat (Prionailurus planiceps), rusty-spotted leopard cat (Prionailurus rubiginosus), fishing cat (Prionailurus viverrinus), Pallas's cat (Otocolobus manul), desert cat (Felis bieti), domestic cat (Felis catus), jungle cat (Felis chaus), African wildcat (Felis lybica), desert cat (Felis margarita), black-footed cat (Felis nigripes), spotted cat (Felis silvestris, etc.

[0063] Freund's adjuvant is the most commonly used adjuvant in animal experiments, and it is divided into incomplete Freund's adjuvant and complete Freund's adjuvant. Incomplete Freund's adjuvant is a mixture of an oil (paraffin oil or vegetable oil) and an emulsifier (lanolin or Tween 80), with a component ratio of 1 to 5:1, which can be determined according to needs, but is usually 2:1. Complete Freund's adjuvant (FCA) is produced by adding BCG (final concentration 2–20 mg / ml) or dead Mycobacterium tuberculosis to incomplete Freund's adjuvant.

[0064] "TTBS": This is a buffer solution commonly used in biological experiments, primarily for washing steps in experiments such as Western blotting and immunohistochemistry (IHC), effectively reducing non-specific binding. It can be prepared using conventional methods in the field, such as dissolving 8.8g of NaCl in an appropriate amount of ddH2O, adding 10mL of Tris-HCl (pH=8.0) and 500μL of Tween 20, and then bringing the volume to 1L. Store at room temperature before use.

[0065] Example 1: Synthesis of AIM protein and preparation of mouse anti-cat AIM protein polyclonal antibody

[0066] rAIM protein was synthesized by GenScript according to the sequence of AIM protein.

[0067] 50 μg of rAIM protein (0.29 mg / mL) was mixed with an equal volume of complete Freund's adjuvant and administered to mice via intraperitoneal injection. The mice were turned over with their heads tilted downwards, and the needle was inserted at a 45-degree angle, close to the midline of the abdomen, injected slowly, and then quickly withdrawn. A booster immunization was administered 14 days after the initial immunization. 100 μl of blood was collected after the initial immunization. 50 μg of rAIM protein was mixed with an equal volume of incomplete Freund's adjuvant and administered to mice via intraperitoneal injection. A third immunization was administered 14 days later, using the same method as the second immunization. Blood was collected 1 mL after enucleation from the mice 14 days later. The blood was incubated at 37°C for 2 hours, centrifuged at 12,000 rpm for 10 minutes, and the serum was collected and stored at -20°C. Using pre-immunization serum as a negative control, the antibody titer of the serum was detected by enzyme-linked immunosorbent assay (ELISA). The titers of the polyclonal antibodies against AIM protein prepared from mice A, B, C, and D were 1:128000, 1:128000, 1:128000, and 1:256000, respectively, and all of them could be used for Western blotting.

[0068] Example 2: Effect of in vitro screening additives on the dissociation of IgM-AIM complex

[0069] Add 20 μL of feline serum to a 0.5 mL EP tube, and add 5 mM of the test sample. For the control group, use ddH2O to make up the difference. Mix well and incubate at 39 °C. After incubation for different times (0-24 h), extract total protein from the feline serum and analyze AIM levels by Western blotting.

[0070] Total protein was extracted from cat serum using the following method: Cat serum incubated for different times was taken out, and the protein concentration in the serum was measured by the BCA method. After calculation, 200 ng of cat serum was taken, and PBS was added to make up to a total volume of 40 μL. Then, 10 μL of 5× loading buffer (non-reducing) was added, mixed well, and 10 μL of the sample was loaded and subjected to immunoblotting detection.

[0071] Immunoblot analysis of AIM levels: Western blotting (WB) was performed using 4%–20% gradient SDS-PAGE gels. 30 μg of total protein sample was added to each well, with 30 ng of rAIM used as a positive control. Transfer was performed at 300 mA at 4°C for 3.5 h. After transfer, the membrane was blocked with 5% skim milk powder for 1 h. Subsequently, the mouse anti-cat AIM polyclonal antibody prepared in Example 1 (1:1500 dilution) was used as the primary antibody and incubated overnight at 4°C. Then, the membrane was incubated with the secondary antibody (HRP (horseradish peroxidase)-labeled goat anti-mouse, 1:5000 dilution) at room temperature for 2 h, followed by washing the NC membrane 6 times for 5 min with TTBS. Finally, the membrane was exposed in a chemiluminescence analyzer, labeled according to protein marker size, and the experimental results were analyzed.

[0072] like Figure 1-2 As shown, through immunoblotting analysis, we found that with the increase of co-incubation time with cat serum, L-cysteine ​​and the combination of L-cysteine ​​+ methionine both showed a promoting effect on the dissociation of IgM-AIM complex at different time points, with the effect being most significant after 24 hours of co-incubation. However, no significant changes were observed when methionine was used alone.

[0073] A concentration gradient experiment was conducted using L-cysteine, L-carnitine, vitamin B1, and cystine to verify their effects on the dissociation of the IgM-AIM complex in feline serum. 20 μL of feline serum was added to a 0.5 mL EP tube, followed by different concentrations of the additives. The control group was supplemented with ddH2O. After mixing, the tubes were incubated at 39°C for 24 h. Following incubation, total protein was extracted from the feline serum using the same method, and AIM levels were analyzed by Western blotting.

[0074] like Figures 3A-3C As shown, the results demonstrate that L-cysteine, L-carnitine, and vitamin B1 can dissociate the IgM-AIM complex in cat serum, but cysteine ​​has no significant effect.

[0075] Example 3: In vivo evaluation of the effect of additives on the dissociation of the IgM-AIM complex

[0076] Healthy experimental cats were selected and administered oral medications (2 mL / group) to the following groups: water, L-cysteine ​​(200 mg), L-carnitine (200 mg), vitamin B1 (1 mg), and an additive combination group (L-cysteine ​​200 mg, L-carnitine 200 mg, vitamin B1 1 mg). Two cats were assigned to each group. In the L-cysteine ​​group, the cats weighed 2.75 kg and 3.0 kg, respectively; in the L-carnitine group, the cats weighed 2.3 kg and 4.3 kg, respectively; in the vitamin B1 group, the cats weighed 3.9 kg and 3.5 kg, respectively; and in the additive combination group, the cats weighed 2.2 kg and 3.8 kg, respectively. Blood samples were collected on days 0, 3, 6, and 9. Total protein was extracted from the cat serum using the same method as described in Example 2, and AIM levels were analyzed by Western blotting to assess AIM protein activation.

[0077] Such as 4A-4C, Figure 5 As shown, the combination of L-cysteine, L-carnitine, vitamin B1, and additives can all promote the dissociation of the IgM-AIM complex in vivo.

[0078] This application verifies through in vitro and in vivo experiments that L-carnitine can promote the dissociation of the IgM-AIM complex in cat serum and activate AIM protein. It can be used to improve renal function and prevent or treat kidney disease in felines. It can be used to prepare compositions for improving renal function and preventing or treating kidney disease in felines, such as prescription diets, foods, food additives, health products, nutritional supplements, etc., and has broad application prospects.

[0079] The scope of protection of this application is not limited to the embodiments described above. Obviously, those skilled in the art can make various modifications and variations to this application without departing from the scope and spirit of the invention. If such modifications and variations fall within the scope of the claims of this application and their equivalents, then the intent of this application also includes such modifications and variations.

[0080] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

[0081] Furthermore, various different implementations of this application can be combined in any way, as long as they do not violate the spirit of this application, they should also be regarded as the content disclosed in this application.

Claims

1. The use of L-carnitine or a physiologically acceptable salt thereof as the sole active ingredient in the preparation of compositions for improving renal function in felines by promoting the dissociation of the IgM-AIM complex; wherein the improvement of renal function is due to the improvement of renal function caused by IgM The inability of the AIM complex to dissociate and the inability of AIM protein to activate leads to abnormal kidney function, kidney dysfunction, kidney deficiency, and kidney damage.

2. The application according to claim 1, characterized in that, The composition was administered to felines via the gastrointestinal tract.

3. The application according to claim 1, characterized in that, The composition was administered parenterally to felines.

4. The application according to claim 1, characterized in that, The composition is administered orally to felines.

5. The application according to any one of claims 1-4, characterized in that, The composition is a prescription diet.

6. The application according to any one of claims 1-4, characterized in that, The composition is a food product.

7. The application according to any one of claims 1-4, characterized in that, The composition is a food additive.

8. The application according to any one of claims 1-4, characterized in that, The composition is a health product.

9. The application according to any one of claims 1-4, characterized in that, The composition is a nutritional supplement.

10. The use of L-carnitine or a physiologically acceptable salt thereof, and L-cysteine ​​or a physiologically acceptable salt thereof as the main active ingredients in the preparation of compositions for improving renal function in felines by promoting the dissociation of the IgM-AIM complex; wherein the improvement of renal function is due to the improvement of renal function caused by IgM The inability of the AIM complex to dissociate and the inability of AIM protein to activate leads to abnormal kidney function, kidney dysfunction, kidney deficiency, and kidney damage.

11. The application according to claim 10, characterized in that, The composition was administered to felines via the gastrointestinal tract.

12. The application according to claim 10, characterized in that, The composition was administered parenterally to felines.

13. The application according to claim 10, characterized in that, The composition is administered orally to felines.

14. The application according to any one of claims 10-13, characterized in that, The composition is a prescription diet.

15. The application according to any one of claims 10-13, characterized in that, The composition is a food product.

16. The application according to any one of claims 10-13, characterized in that, The composition is a food additive.

17. The application according to any one of claims 10-13, characterized in that, The composition is a health product.

18. The application according to any one of claims 10-13, characterized in that, The composition is a nutritional supplement.

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