JP7232453B2 - Diabetic retinopathy, cataract and/or nephropathy model laboratory animal - Google Patents

Description

The present invention relates to diabetic retinopathy, cataract and/or nephropathy model experimental animals.

Diabetic retinopathy is one of the three major complications of diabetes and a leading cause of blindness in adults. It is possible to confirm the progress of symptoms and the effect of treatment by clinical diagnosis and pathological autopsy. However, the pathological characteristics of diabetic retinopathy and the mechanism of progression have not been elucidated. Experimental animals exhibiting similar symptoms are very important for deep understanding of diabetic retinopathy at the molecular and cellular level.
Diabetic nephropathy is also one of the three major complications, and is a typical secondary glomerular disease. Since 1998, it has been the number one disease for which chronic dialysis therapy is introduced in Japan. As with diabetic retinopathy, there are many unclear points about the pathophysiological characteristics and progression mechanisms, and a deep understanding at the molecular and cellular level is necessary for prevention and treatment.
Representative experimental animals showing partial features of type II diabetic nephropathy include C57BL/6J mice with pancreatic destruction by streptozotocin treatment and FVB mice genetically rendered type I and type II diabetic. Using fragile db/db mice and Black and tan, branchyury (BTBR) mice with a genetic background of insulin resistance, these diabetic-characterized mice were genetically engineered to be predisposed to hypertension. Mice or mice in which renal function is reduced by surgically removing one of a pair of kidneys and the load on one kidney is increased (Betz & Conway, Recent advances in animal models of diabetic nephropathy Nephron Experimental Nephropathy 2014;126, 191-195). The type II diabetic nephropathy model mouse described above exhibits early lesions of human diabetic nephropathy and is an experimental animal that can contribute to the elucidation of the cause of lesions. There is a clearly different process from humans, such as treatment, and there is a problem that it is different from lifestyle-related diseases.
In recent years, the 3Rs (Reduce number of animals used [Reduce], Refinement [Refinement], Utilization of alternative methods [Replacement]) in animal experiments have been widely recognized as international principles of laboratory animal welfare, and the use of mammals is strictly regulated. I started. As an alternative method, the use of small fish is recommended. Especially in the field of industry such as drug development, the importance of small fish is increasing. For example, the whole-animal drug screening method has begun to spread as a next-generation method of compound screening method for selecting new drug candidate compounds, and zebrafish is used for it.
There is a report on a retinopathy model using zebrafish (Non-Patent Document 1). Zebrafish and human eyes have a great similarity in structure, and are widely used in research on eye development and visual impairment (Non-Patent Document 2). It is a small fish with a body length of 3 cm or less, and has the advantage of being less expensive and less space-saving than laboratory rodents. Genome editing and genetic recombination can be performed relatively easily (Non-Patent Document 3), and it is expected that not only the morphological analysis of diabetic retinopathy but also the use of related mutant strains will contribute to the identification of the causative gene. .
Similarly, there is a report on diabetic nephropathy using zebrafish (Olsen AS, Sarras MP, Intine RV. Limb regeneration is impaired in an adult zebrafish model of diabetes mellitus Wound Repair Regen 2010, 18(5):532- 542.). Fish, including zebrafish and medaka, also have renal structures similar to those of humans and rodents, and are used to create model organisms and to analyze the functions of genes related to nephropathy (Seman NA, He B, Ojala JRM, Mohamud WNW, Ostenson CG, Brismar K, Gu HF. Genetic and biological effects of sodium-chloride cotranspoter (SLC12A3) in diabetic nephropathy. American Journal of Nephropathy 201440:408-416).
Medaka is currently a small experimental fish that is as useful as zebrafish, and genome editing and genetic recombination can be performed relatively easily (Non-Patent Document 3).
However, no experimental animal model exhibits all of the symptoms of diabetic retinopathy and/or cataract or nephropathy in humans.
By the way, the leptin receptor is the receptor for leptin, an appetite suppressing hormone. It was identified by Tartaglia et al. and Lee et al. The present inventors produced leptin receptor-deficient (LepRKO) medaka by the TILLING method, and reported that LepRKO medaka has a higher food intake and grows faster than wild-type medaka (Non-Patent Documents 6, 7, Patent document 1). In addition, it was reported that abnormalities in glucose metabolism were exhibited, and fasting hyperglycemia was exhibited in breeding with a specific feed amount (Table 1) (Non-Patent Documents 8, 9, 10, 11, 12).
In a report of a retinopathy model using zebrafish, immersing zebrafish in a high-concentration glucose solution can maintain the whole body in a hyperglycemic state and induce thinning of the intraretinal granular layer (Non-Patent Document 1). . However, no other symptoms were observed.
There is a report that medaka showed fasting hyperglycemia after feeding with a high-fat diet, but there is no report of diabetes-related retinopathy and cataract (Non-Patent Document 13).

JP 2014-147378 A

Gleeson M, et al., Acta Diabetologica, 44(3):157-163, 2007 Goldsmith P and Harris WA, Seminars in Cell and Developmental Biology 14(1):11-18, 2003 Kawahara A, et al., Chapter 8 in Targeted genome editing using site-specific nucleases, 119-131, 2014 Tartaglia LA, et al., Cell, 83: 1263-1271, 1995 Lee GH, et al., Nature, 379:632-635,1996 Shinichi Kamata, Journal of Comparative Endocrinology, 40(152):101～103, 2014 Chisada S, et al., Gen Comp Endocrinol, 195: 9-20, 2014 Chisada S, et al., Abstracts of 7th Aquatic Animal Models for Human Disease Conference, 152 page, 2014 Chisada S, et al., Abstracts of the 20th Small Fish Research Meeting, 42 page, 2014 Chisada S, et al., Abstracts of the 21st Small Fish Research Meeting, 33 page, 2015 Chisada S, et al., Abstracts of 9th European zebrafish meeting, P2-O32, 2015 Chisada S, et al., Abstracts of Aquatic model organisms for human disease and toxicology research, 22 pages, 2016 Mastumoto et al., Disease Models & Mechanisms, 3:431-440, 2010

Under these circumstances, development of experimental animal models for diabetic retinopathy, cataract, nephropathy, etc. has been desired.

As a result of intensive studies in order to solve the above problems, the present inventors have found that diabetic retinopathy, cataract and/or nephropathy can be caused by breeding small experimental fish lacking leptin receptors according to a predetermined feeding method. We have succeeded in producing a model experimental animal, and have completed the present invention.

That is, the present invention is as follows.
(1) Experimental small fish with a functional deficiency in leptin receptors were treated at any time from hatching to 6 weeks after hatching to 20 weeks or more after hatching, and the age after hatching and the amount of food fed. A method for causing diabetic retinopathy, cataract and/or nephropathy in the small experimental fish, characterized by feeding and rearing such that the ratio is within the range of the following values.
Feeding amount (mg/tail/day)/age in weeks after hatching = 0.69 to 1.96
(2) The method according to (1), wherein the ratio of the post-hatch age in weeks to the feed amount is within the following range.
Feeding amount (mg/tail/day)/age in weeks after hatching = 1.0-1.96 (up to 19 weeks after hatching, excluding 4 weeks after hatching), 0.69-1.34 (4 weeks after hatching and 20 weeks after hatching) weeks of age and later)
(3) The method according to (1) or (2), wherein the leptin receptor functional deficiency is a leptin receptor deficiency homozygote.
(4) The method according to any one of (1) to (3), wherein the small experimental fish is killifish or zebrafish.
(5) A fish model of diabetic retinopathy, cataract and/or nephropathy prepared by the method according to any one of (1) to (4).
(6) Experimental small fish with a functional defect in the leptin receptor are treated at any time from hatching to 6 weeks after hatching to 20 weeks or more after hatching, and the age after hatching and the amount of food fed A method for producing diabetic retinopathy, cataract and/or nephropathy model fish, characterized by feeding and breeding so that the ratio is within the range of the following values.
Feeding amount (mg/tail/day)/age in weeks after hatching = 0.69 to 1.96
(7) The method according to (6), wherein the ratio of the post-hatch age in weeks to the feed amount is within the following range.
Feeding amount (mg/tail/day)/age in weeks after hatching = 1.0-1.96 (up to 19 weeks after hatching, excluding 4 weeks after hatching), 0.69-1.34 (4 weeks after hatching and 20 weeks after hatching) weeks of age and later)
(8) A method for screening therapeutic agents for diabetic retinopathy, cataract and/or nephropathy, which comprises contacting the model fish of (5) with a candidate substance.

The present invention provides experimental animals for diabetic retinopathy, cataract and/or nephropathy models (models of diabetic retinopathy, cataract, nephropathy, or a combination thereof). Using the experimental animals of the present invention (for example, 20-week-old to 28-week-old LepRKO medaka fish), it is possible to perform a large number of drug screenings in a small space and at low cost.
The present invention includes providing medaka fish exhibiting type II diabetic retinopathy, cataract and/or nephropathy. Type II diabetic retinopathy medaka has findings of partial diabetic retinopathy recognized in humans as well as existing model animals. However, it exhibits earlier and more pronounced retinopathy, cataract and/or nephropathy than existing animal models. And it is the first medaka with histopathological characteristics of type II diabetic retinopathy, cataract and/or nephropathy using small fish. With the present invention, it is possible to overcome the 3Rs of animal experiments, which are widely recognized as international principles of laboratory animal welfare. have great significance.

FIG. 1 shows specific symptom-inducing feeding amounts, non-symptom-inducing feeding amounts, and average body weights at each age. Bold line: specific feeding dose (average) that induces symptoms, corresponding to Table 1. Shaded area: lower and upper dose range for a particular feeding dose that induces symptoms. Gray area: feeding dose that did not induce symptoms. Dashed line: body weight values when fed a specific symptom-inducing food intake (mean). FIG. 2 shows fasting blood glucose levels of LepRKO medaka and wild-type medaka bred by the method of the present invention. Black columns: wild-type medaka, white columns: LepRKO medaka. *p<0.001 FIG. 2 shows plasma insulin concentrations in LepRKO medaka and wild-type medaka raised by the method of the present invention 1 hour after feeding. Black columns: wild-type medaka, white columns: LepRKO medaka. *p<0.001 It is a photograph of LepRKO medaka with an opaque lens observed at 28 weeks of age after hatching. A: Wild-type medaka, B: LepRKO medaka. FIG. 10 is a diagram showing a histological observation image of the lens of LepRKO medaka having an opaque lens. A1, A2: wild-type medaka, B1, B2: LepRKO medaka, asterisk: edema, black ▲: Morgani corpuscle, scale of A1 and B1: 100 um, scale of A2 and B2: 50 um. FIG. 10 is a diagram showing a histological observation image of the retina of LepRKO medaka with an opaque lens. A: wild-type medaka, B: LepRKO medaka, a: retinal pigment epithelial cell layer, b: melanin pigment layer, c: photoreceptor layer, d: outer nuclear layer, e: outer reticular layer, f: inner nuclear layer, g : inner plexiform layer, h: optic nerve cell layer, i: optic nerve fiber, asterisk: indicates capillary congestion. Fig. 1 shows blood creatinine concentrations of LepRKO medaka and wild-type medaka in breeding with specific feed amounts (Table 1, Fig. 1). Black columns: wild type, white columns: LepRKO medaka. Differences in a are p<0.05 by Student's t-test, and differences in b are p<0.01 by Mann-Whitney U-test. 20-week-old kidney histological images of LepRKO medaka and wild-type medaka bred with specific feed amounts (Table 1, FIG. 1) are shown. A: Wild-type medaka, B: LepRKO medaka. The scale indicates 20um. 30-week-old kidney histological images of LepRKO medaka and wild-type medaka bred with specific feed amounts (Table 1, FIG. 1) are shown. A: wild-type medaka, B and C: LepRKO medaka. Black ▴ in FIG. B: glomerular capillary space, asterisk in FIG. B: dilation of afferent and efferent arteries, arrow in FIG. 9C: hyperplasia of mesangial matrix, black ▴ in FIG. 9C: hyaline-like substance, scale indicates 20 μm. 40-week-old kidney histological images of LepRKO medaka and wild-type medaka bred with specific feed amounts (Table 1, FIG. 1) are shown. A: Wild-type medaka, B: LepRKO medaka. FIG. 10B, asterisk: dilation of Bowman's capsule space, FIG. 10B, black triangle: dilation of glomerular capillary space, scale indicates 20 um.

1. Overview The present inventors bred leptin receptor-deficient medaka with a specific feeding amount, and measured the blood glucose level and insulin concentration of the medaka, thereby showing that the medaka has type II diabetes-like symptoms. Furthermore, the inventors have found that the medaka fish are useful as diabetic retinopathy/cataract model animals and diabetic nephropathy model animals. The present invention is an invention completed based on these findings.

An object of the present invention is to provide a novel pathological model experimental animal that reproduces retinopathy, cataract and/or nephropathy from type II diabetes, a method for producing the same, and a method for using the novel pathological model experimental animal. The present inventors have conducted intensive studies in view of the above purpose, and as a result, LepRKO medaka under breeding with a specific feeding amount (Table 1, Figure 1), fasting hyperglycemia, and retinopathy, cataract and / or maintained histopathological features of nephropathy.

Surprisingly, compared with experimental animals with existing symptoms of type II diabetic retinopathy, the lesions obtained in LepRKO medaka were induced relatively early and the lesions were remarkable. rice field. The lesion is characterized by histopathologic features such as marked congestion of blood vessels running to the optic nerve fiber layer, consequent compression of the retinal parenchyma, severe retinal thinning, retinal detachment, degeneration of lens fibers, and formation of an opaque lens. And so on. For example, in the present invention, LepRKO medaka developed lens degeneration (cataract) at 28 weeks of age. This is clearly early onset compared to existing model animals. For example, previous reports have shown that model rodents undergo lens degeneration at 40 and 76 weeks of age.
Regarding renal lesions, lesions are not observed particularly early compared to existing model rodents. An example of early diabetic nephropathy lesions in rodents is the Black and tan, branchyury (BTBR) mouse, in which lesions are observed at 22 weeks of age (Betz & Conway, Recent advances in animal models). of diabetic nephropathy. Nephron Experimental Nephropathy 2014;126, 191-195). However, many existing model rodents, including BTBR mice, differ from humans by exhibiting renal lesions despite normal blood creatinine levels. In humans, when diabetic nephropathy occurs, renal function usually declines, creatinine cannot be excreted from the body, and the blood creatinine concentration increases. LepRKO medaka has the characteristics of type II diabetes, and the blood creatinine level rises from 20 weeks after hatching, and no significant changes in kidneys are observed. , the blood creatinine concentration is significantly increased, and lesions characteristic of diabetic nephropathy observed in humans and model rodents are also observed. In addition, existing model rodents need to be predisposed for diabetes and genetically engineered for hypertension. On the other hand, in LepRKO medaka, dilation of glomerular capillary cavities, Bowman's capsule, etc., presumed to be caused by hypertension, is observed only at specific feeding amounts (Table 1, Fig. 1). In addition, in the present invention, small fish functionally deficient in leptin receptors can be bred only with specific feeding amounts (Table 1, Fig. 1) without drugs, surgical treatment or genetic manipulation. Fasting hyperglycemia and histopathological features of retinopathy, cataract and/or nephropathy can be preserved.

2. Production of cataract, retinopathy and/or nephropathy model fish
In the method of the present invention, small laboratory fish having a functional defect in the leptin receptor are bred. The term "leptin receptor functional deficiency" means a state in which the leptin receptor is unable to fully exhibit its original normal function, and includes knockout of the leptin receptor. Experimental small fish with a functional defect in the leptin receptor are gene-mutated fish accompanied by genomic DNA mutation or recombination, or genetically-modified fish, regardless of the presence or absence of other gene mutations or modifications.

The target fish for breeding, ie fish with a functional defect in the leptin receptor, are small laboratory fish, for example killifish, zebrafish or goldfish. In the present invention, the fish to be used is not particularly limited as long as it is a small experimental fish having a functional defect in the leptin receptor. is knocked out, and the like.

Techniques for producing medaka with knockout leptin receptors are known (Chisada S, et al., Gen Comp Endocrinol, 195: 9-20, 2014, Taniguchi et al, Genome Biol, 7(12):R116, 2006), and medaka produced by this method can be used. However, it is not limited to these known methods.
In addition, zebrafish in which the leptin receptor is knocked out can be relatively easily obtained by using genome editing technology (Kawahara A, et al., Chapter 8 in Targeted genome editing using site-specific nucleases, 119-131, 2014). can be made.
The leptin receptor-deficient fish thus obtained are reared at specific feed rates (Table 1, Figure 1) with defined ranges and population densities.

The "predetermined range" is a range of 20% or more and 20% or less of the feeding amount (mg) shown in Table 1 at each week of age after hatching. It is characterized by feeding and rearing so that the relationship (ratio) between the post-hatch age in weeks and the feeding amount satisfies the following formula I.
Feeding amount (mg/tail/day)/age in weeks after hatch = 0.69-1.96 (I)
The range that satisfies the above formula I is the shaded range in FIG. 1, and this value is an index of the amount that maintains a sufficient swelling of the belly.
In the present invention, the amount of feed can be reduced at any one week of age up to 19 weeks of age, or at 2 to 4 weeks of age up to 19 weeks of age. The ratio of the amount of food fed to the age after hatching when weight is reduced (the value of Formula I) is between 0.69 and 1.34.
The age at which the weight is to be reduced can be appropriately determined in consideration of factors such as swelling of the stomach of the fish, longevity of the fish, particle size of the food, and outflow of the fed food from the tank. In order to keep the belly of the medaka sufficiently swollen, it is necessary to feed the medaka with a larger amount of food with a small particle size than with a food with a large particle size. This is because if the particle size is small, it is predicted that it will float on the surface of the water for a long time and is likely to flow out (the water tank is a circulating tank), or it will be digested quickly (feces will come out quickly). be. Feeding that satisfies the above ratio means that the medaka continues to be fed with "an amount that maintains a sufficient swelling of the belly" even if the weight is reduced.
For example, at 4 weeks old and 8 weeks after hatching, we started using food with a larger particle size than the previous week, so at this age (4 weeks old, 8 weeks old, or both weeks old), the above ratio be in the range of 0.69 to 1.34. Similarly, 12-week-old, 13-week-old and 20-week-old and beyond can be considered in the same manner as 4-week-old and 8-week-old.
Therefore, in a preferred embodiment of the present invention, until the age of 19 weeks after hatching, the feeding is basically carried out so that the value of formula I is 1.0 to 1.96, but 4 weeks, 8 weeks and 12 weeks of age after hatching. and at least one of 13 weeks of age, and after 20 weeks of age, feed to 0.69-1.34. Preferably, the value of Formula I is essentially between 1.0 and 1.96, but between 0.69 and 1.34 after 4 weeks of age and 20 weeks after hatch.

Feeding can be started any time from hatching to 6 weeks after hatching, and feeding can be finished any time after 20 weeks after hatching.
The specific feeding amount is, for example, 8.4 mg±1.2 mg per fish per day at 6 weeks of age, and 20.0 mg±4.0 mg per fish per day at 16 weeks of age. The feeding amount at 20 weeks of age is 22.3±4.3 mg per fish per day, and at 28 weeks of age it is 23.3±2.7 mg. After 29 weeks of age, feed according to formula I (feed amount (mg/tail/day)/age in weeks after hatching = 0.69 to 1.34).

Feeding should be divided into 4 or more times (for example, 4 to 6 times) per day. After one feeding, wait at least 1 hour.
Individual densities range from 1 to 4 fish per liter.
The breeding environment other than the above is the same as the general method. mg NO2-/L or less, 20 mg NO3-/L or less, hardness 20-100 mg CaCO3/L or less, use of a collection tank with circulation filtration, light/dark period = 14 hours/10 hours (Naruse et al. al, Exp Anim, 59(1):13-23, 2010).
The feed may be similar to general fish feed containing protein, lipid, dietary fiber, coarse ash, Ca and phosphorus. In Table 1 and Figure 1, Otohime A [protein 53% or more, lipid 8% or more, dietary fiber 3% or less, crude ash content 16% or less, Ca 2.3% or more, phosphorus 1.5% or more] and B1, B2 [protein 50% or more, 10% or more fat, 3% or less dietary fiber, 16% or less crude ash, 2.0% or more Ca, 1.5% or more phosphorus] (Marubeni Nisshin Feed Co., Ltd.).

Feeding in this manner causes hyperglycemia, retinopathy, cataracts, nephropathy, or a combination of two or more of these diseases. The onset time of these symptoms is not limited.
In the case of medaka, it is difficult to reproduce retinopathy, cataract, and/or nephropathy from type II diabetes if, for example, the following conditions a to e apply, so these conditions should be avoided.

ah. When the amount of feeding below the hatched area in FIG. 1 is continued.
stomach. If the amount of feeding in the hatched area in FIG. 1 (the amount outside the range of 0.69 to 1.96 for formula I) is not started within 6 weeks of age after hatching.
hare. Even if the amount of the shaded area in Fig. 1 (the amount within the range of 0.69 to 1.96 in formula I) was started within 6 weeks after hatching, it was not continued until after 20 weeks after hatching. case.
workman. When breeding at an individual density of 5 fish / 1L or more.
E. If the daily feeding amount is fed within 3 times.

In the case of "a-e" above, the leptin receptor-deficient medaka shows poor growth due to insufficient food intake and density effects, and does not show diabetic symptoms due to overeating. That is, it does not show diabetic retinopathy, cataracts and/or nephropathy. In the case of "E", the food remains excessively on the bottom of the water, the deterioration of the water quality results in poor growth, and similarly no diabetic retinopathy, cataract and/or nephropathy.

By selecting experimental small fish with a functional deficiency in leptin receptors as model experimental animals that reproduce retinopathy, cataract and/or nephropathy from type II diabetes, specific feeding amounts (Table 1, Figure 1) Breeding alone makes it possible to induce marked histopathological characteristics of diabetic retinopathy and/or cataract relatively early compared to experimental animals with existing symptoms of type II diabetic retinopathy. and induces histopathological features and a decline in renal function not seen in most rodent models, not later than in experimental animals with pre-existing type II diabetic nephropathy symptoms. can do.

Unlike mammals that are functionally deficient in leptin receptors, even homozygous mutants have reproductive ability, making it possible to maintain stable strains and secure individuals. It is easy to secure enough individuals for statistical processing, even if individual differences occur in spite of being a strain with a uniform genetic background. In addition, compared with model rodents of diabetic retinopathy, etc., it exhibits remarkable histopathological characteristics relatively early. Therefore, it is possible to analyze histopathological characteristics over time relatively early. For example, by reproducing retinopathy, cataract and/or nephropathy from type II diabetes using LepRKO medaka and following the progression of lesions over time, elucidation of the pathophysiological mechanism can be achieved in a short period of time (e.g., within 28 weeks of age). And compared with experimental animals with existing type II diabetic nephropathy symptoms, at a time not particularly late, without special drug treatment and genetic manipulation, a specific feeding amount (Table 1, Figure 1) can induce histopathological features and a decline in renal function absent in many model rodents. For example, LepRKO medaka that reproduces retinopathy, cataract and/or nephropathy from type II diabetes, or 20-week-old to 28-week-old (retinopathy and/or cataract) bred with a specific feeding amount (Table 1, Figure 1) Using multiple LepRKO medaka at 20 to 40 weeks of age (in the case of nephropathy), it is possible to perform drug screening in a smaller space and at a lower cost than rodents.

The model fish of the present invention exhibits remarkable histopathological characteristics at a relatively early stage compared to diabetic retinopathy model rodents and the like. Therefore, LepRKO medaka that reproduces retinopathy, cataract and / or nephropathy from type II diabetes, or LepRKO medaka aged 20 weeks to 28 weeks bred with a specific feeding amount (Table 1, Figure 1) By using the number of tails, it is possible to analyze histopathological characteristics over time relatively early, and to perform a large number of drug screenings in a small space and at a low cost compared to rodents. In existing diabetic nephropathy model rodents, multiple treatments are required due to the concurrent nature of diabetes and hypertension. 1, Fig. 1), it is possible to follow the progression of lesions over time. Therefore, in humans, it is possible to analyze situations similar to the progression of lifestyle-related diseases.

3. Screening Method The leptin receptor-deficient fish bred by the method of the present invention is a model fish for diabetic retinopathy, cataract and/or nephropathy. Therefore, the model fish can be used to screen therapeutic agents for diabetic retinopathy, cataract and/or nephropathy.
Therefore, the present invention provides a screening method for therapeutic agents for diabetic retinopathy, cataract and/or nephropathy, which comprises contacting or administering a candidate substance to the model fish.

It was found that the model fish of the present invention exhibited phenotypes (disease symptoms) of diabetic retinopathy, cataract, nephropathy, or a combination thereof when fed with a predetermined amount of food for a predetermined period of time. Based on this knowledge, if any substance can be confirmed to have the effect of alleviating or alleviating diabetic retinopathy, cataract and/or nephropathy in the model fish, the substance is an anti-retinopathy drug, an anti-cataract drug, and/or can be used as an anti-nephropathic agent.
Candidate substances are not particularly limited, and may be existing drugs (e.g., existing retinopathy drugs, existing cataract drugs, existing nephropathy drugs), as well as peptides, low-molecular-weight compounds, high-molecular-weight compounds, and salts thereof. Alternatively, it may be in any form such as a precursor.

The screening of the present invention specifically includes the following steps.
(a) a step of contacting the model fish of the present invention with a candidate substance (b) a step of examining reduction of diabetic retinopathy, cataract and/or nephropathy in the contacted fish There is an embodiment in which a candidate substance is administered to a model fish. Administration can be oral or parenteral. That is, the route of administration of the candidate substance is not particularly limited as long as it is a route commonly used for administration of drugs, and examples thereof include oral, transdermal, and injection routes. "Contact" also includes a mode in which the candidate substance is absorbed from the body surface of the fish (percutaneous absorption) by adding the candidate substance to an aquarium containing the model fish of the present invention.

Items to be inspected in step (b) are at least one of the following (i) to (xv).
(i) Blood sugar level
(ii) blood insulin concentration
(iii) Vacuolization/liquefaction of lens fibers
(iv) Formation of lens fiber edema
(v) degeneration of lens fibers (including formation of Morganey bodies);
(vi) congestion of retinal capillaries and consequent retinal compression;
(vii) thinning in the retinal pigment epithelial layer, photoreceptor layer, outer nuclear layer, outer reticular layer, inner nuclear layer, inner reticular layer or nerve fiber layer;
(viii) decreased number of optic nerve cells
(ix) dilatation of the glomerular capillary lumen and afferent and efferent arteries
(x) glomerular swelling
(xi) growth of mesangial matrix
(xii) retention of hyaline material within the glomerular capillary lumen;
(xiii) Dilation of Bowman's space
(xiv) glomerular loop atrophy
(xv) blood creatinine concentration

A candidate substance can be selected as an anti-retinopathic agent, an anti-cataract agent and/or an anti-nephropathic agent if at least one of the above mentioned items is ameliorated by contact with the candidate substance.

Examples Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples.

[Reference example 1]
Generation of leptin receptor-deficient medaka fish
LepRKO medaka was produced by a known method (Chisada S, et al., Gen Comp Endocrinol, 195: 9-20, 2014).

Breeding of leptin receptor-deficient medaka with specific feeding amounts (Table 1, Fig. 1) 1. Method
Wild-type medaka (Kyoto-Cab strain) was used as a control group together with LepRKO medaka. These medaka were reared in an environment adjusted to a water temperature of 26±1°C and a long day condition of 14 hours light and 10 hours dark. LepRKO medaka has been reported to consume 1.5-1.7 times more food per unit time than wild-type medaka (Chisada S, et al., Gen Comp Endocrinol, 195: 9-20, 2014). . Therefore, the amount shown in Table 1 was fed to LepRKO medaka, and 66% of the amount was fed to wild-type medaka. The amount of food to be fed at one time is the amount that the medaka in the tank can eat within 30 minutes. Therefore, when feeding the amount of feed per day shown in Table 1 and Fig. 1, the amount was divided into 5 to 7 times. The bait used is shown in Table 2.

Blood glucose level of leptin receptor-deficient medaka in breeding with specific feeding amount (Table 1, Fig. 1) 1. Method
LepRKO medaka and wild-type medaka were used. Normally, LepRKO medaka show fasting hyperglycemia within 5-12 weeks after hatching when reared with specific feeding amounts (Table 1, Fig. 1). The fasting blood glucose level is the blood glucose level measured after 18 hours of fasting of medaka raised with a specific feeding amount (Table 1, Fig. 1). In this study, blood glucose levels were measured at 10 weeks after hatching with macroscopically normal lenses and at 28 weeks of age including individuals with opaque lenses.

2. Results At 10 weeks after hatching, there was no significant difference in fasting blood glucose levels between LepRKO medaka and wild-type medaka. high and the difference was statistically significant (Fig. 2).

Plasma insulin concentration of leptin receptor-deficient medaka in rearing with a specific feeding amount (Table 1, Fig. 1) 1. Method "Blood glucose level of leptin receptor-deficient medaka raised with a specific amount of food (Table 1, Figure 1)" Using medaka used for measurement and raised under the same conditions at the same time, plasma 1 hour after feeding Insulin concentration was measured based on a known method (Kubo et al., Exp Eye Res, 73:375-381, 2001). However, 10-week-old medaka are too small for blood collection, so only plasma insulin concentrations at 28-week-old were measured.

2. Results The plasma insulin concentration of LepRKO medaka at 28 weeks of age after hatching 1 hour after feeding was clearly lower than that of wild-type medaka (Fig. 3). Therefore, considering the results obtained in Example 2, it was clarified that LepRKO medaka shows hyperglycemia due to insulin secretory deficiency, and that these symptoms are common to type II diabetes-like symptoms.

Histological analysis of the eyes of leptin receptor-deficient medaka fish reared with specific feeding amounts (Table 1, Fig. 1) 1. Method "blood glucose level of leptin receptor-deficient medaka in breeding with a specific feeding amount (Table 1, Figure 1)" and "blood creatinine in leptin receptor-deficient medaka in breeding with a specific feeding amount (Table 1, Figure 1) Using the medaka used for the concentration measurement and the medaka raised under the same conditions at the same time, after fixing with Bouin's fixative and 10% neutral buffered formalin fixative, paraffin sections were made by a conventional method, and tissues were stained with hematoxylin and eosin. scientifically observed.

2. Results Regarding Eyes In the same aquarium where LepRKO medaka showing fasting hyperglycemia and low plasma insulin concentration 1 hour after feeding appeared, medaka with cloudy lens appeared (Fig. 4). As a result of histological analysis of the eyes, marked lens degeneration including vacuolization/fragmentation of lens fibers, formation of edema (asterisks in FIG. 5), and formation of Morganey bodies (black triangles in FIG. 5) was observed. In addition, congestion of retinal capillaries was observed in the retinal optic nerve layer (asterisk in FIG. 6), and retinal compression due to the luminally dilated blood vessels was observed. In addition, the retinal pigment epithelial layer (Fig. 6a), photoreceptor layer (Fig. 6c), outer nuclear layer (Fig. 6d), outer reticular layer (Fig. 6e), inner nuclear layer (Fig. 6f), inner reticular layer (Fig. 6g) , thinning was seen in the nerve fiber layer (Fig. 6i), and the number of optic nerve cells (Fig. 6h) was also reduced.

Congestion of retinal capillaries and consequent retinal compression is an important finding observed in human diabetic retinopathy. In this example, LepRKO medaka that showed fasting hyperglycemia and a low plasma insulin concentration after 1 hour of feeding appeared. Out of 6 medaka fish derived from the same tank, 2 cases had degeneration of the lens, and all had capillary vessels. Congestion of the capillaries and thinning of the retina were observed.
3. 20-week-old, 30-week-old, and 40-week-old LepRKO medaka were collected from the same tank in which LepRKO medaka with fasting hyperglycemia and low plasma insulin concentration 1 hour after feeding appeared, Histological analysis of the kidney was performed. At 20 weeks of age, no significant change was observed in the renal glomeruli of LepRKO medaka compared to wild-type medaka (Fig. 8). At 30 weeks of age, glomerular capillary lumen (Fig. 9B, black ▴) and expansion of afferent and efferent arteries (Fig. 9B, asterisk) were observed in the kidney of LepRKO medaka. In addition, swelling of the glomerulus (Fig. 9C), hyperplasia of the mesangial matrix (Fig. 9C, arrow), and hyaline-like substance retention in the glomerular capillary lumen (Fig. 9C, black ▴) were observed. At 40 weeks of age, it was observed that lesions in LepRKO medaka progressed from 30 weeks of age. For example, dilation of Bowman's space (Fig. 10B, asterisk), dilation of the glomerular capillary lumen (Fig. 10B, black triangles), and atrophy of the glomerular loop (left glomerulus in Fig. 10B). These are key findings observed in human diabetic nephropathy. In this example, 5 out of 6 medaka fish derived from the same tank in which LepRKO medaka fish that showed fasting hyperglycemia and low plasma insulin concentration after 1 hour of feeding appeared, glomerular capillary space, Bowman's space In one case, hyperplasia of the mesangial matrix and hyaline-like substance retention in the lumen of the glomerular capillaries were observed. In humans, the former is responsible for postdiabetic hypertension and the latter is a precursor to postdiabetic neovascular proliferation. Therefore, it can be considered that diabetic nephropathy was induced in medaka in the order of diabetes, hypertension, and proliferation of new blood vessels. Moreover, these histological analysis results are accompanied by an increase in blood creatinine concentration.

Blood creatinine concentration of leptin receptor-deficient medaka in breeding with specific feeding amount (Table 1, Fig. 1) 1. Method LepRKO medaka and wild-type medaka at 20 and 40 weeks of age after hatching were used. Blood collection was measured using the blood obtained after fasting for 18 hours from medaka raised with specific feeding amounts (Table 1, Fig. 1). The medaka after blood collection was subjected to histological analysis of the kidney according to the method of Example 4.
2. Results At 20 and 40 weeks after hatching, the difference in mean blood creatinine concentration between LepRKO medaka and wild-type medaka was statistically significant, and the difference widened from 20 to 40 weeks after hatching. (Fig. 7).

Claims (6)

The relationship between the age after hatching and the amount of food supplied satisfies the following formula at any time from hatching to 6 weeks after hatching to 20 weeks after hatching in medaka with a functional deficiency of leptin receptors. A method for causing diabetic retinopathy, cataract and/or nephropathy in the medaka , characterized by feeding and rearing as described above.
Feeding amount (mg/tail/day)/age in weeks after hatching = 0.69 to 1.96 2. The method according to claim 1, wherein the relationship between the age after hatching and the feeding amount satisfies the following formula.
Feeding amount (mg/tail/day)/age in weeks after hatching = 1.0-1.96 (up to 19 weeks after hatching, excluding 4 weeks after hatching), 0.69-1.34 (4 weeks after hatching and 20 weeks after hatching) weeks of age and later) 3. The method of claim 1 or 2, wherein the leptin receptor functional deficiency is a leptin receptor deficiency homozygote. The relationship between the age after hatching and the amount of food supplied satisfies the following formula at any time from hatching to 6 weeks after hatching to 20 weeks after hatching in medaka with a functional deficiency of leptin receptors. A method for producing diabetic retinopathy, cataract and/or nephropathy model medaka , characterized by feeding and rearing as follows.
Feeding amount (mg/tail/day)/age in weeks after hatching = 0.69 to 1.96 5. The method according to claim 4 , wherein the relationship between the age after hatching and the feeding amount satisfies the following formula.
Feeding amount (mg/tail/day)/age in weeks after hatching = 1.0-1.96 (up to 19 weeks after hatching, excluding 4 weeks after hatching), 0.69-1.34 (4 weeks after hatching and 20 weeks after hatching) weeks of age and later) Medaka with a functional defect in the leptin receptor were tested at any time from hatching to 6 weeks after hatching to 20 weeks or more after hatching, and the relationship between the age after hatching and the amount of feed was calculated using the following formula:
Feeding amount (mg/tail/day)/age in weeks after hatching = 0.69 to 1.96
A diabetic retinopathy, cataract and/or nephropathy model medaka is produced by feeding and rearing so as to meet the requirements, and the candidate substance is brought into contact with the medaka . A screening method for therapeutic agents for nephropathy.

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