Construction method and application of animal model of paternal origin renal proximal tubule dysfunction

Abstract

The application discloses a method for constructing a paternal renal proximal tubule dysfunction animal model and application thereof. The paternal renal proximal tubule dysfunction animal model is obtained by the following steps: continuously giving 60 mg / kg·d caffeine intragastrically to male rats in the breeding age for 8 weeks every day, then impregnating the male rats with normal female rats, obtaining offspring, weaning the offspring at 4 weeks after birth, and feeding the offspring until 32 weeks, wherein proximal tubule dysfunction appears in the offspring, is manifested by disorder of hematuria biochemical indexes, significant decrease of proximal tubule structure and key gene of transport, and obvious increase of specific damage indexes, and adult offspring proximal tubule epithelial cells fall off, and transparent, granular or cellular cast can be seen in the lumen. The model established by the application is novel, reliable and simple, and is more in line with the genetic acquired process of human beings, and has important significance for researching the occurrence mechanism of paternal renal proximal tubule dysfunction and determining a clinical early warning and intervention target.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, specifically to a method for constructing an animal model of paternal renal proximal tubule dysfunction and its application. Background Technology

[0002] The proximal tubule (PT) is the most abundant cell type in the kidney. Its main function is to reabsorb the vast majority of salts and water, as well as solutes. The PT has a high energy requirement to maintain transport, which may explain why it is the most vulnerable segment of the renal tubule. Impaired proximal tubule function can lead to significant loss of fluids, electrolytes, and low molecular weight nutrients. This dysfunction, known since the early 20th century as renal Fanconi syndrome (FRTS), most commonly occurs in children and is part of a multisystem metabolic disorder [1]. Due to the inability to reabsorb key molecules filtered by the kidneys, such as bicarbonate, calcium, or phosphate, dysfunction of this segment of the renal tubule leads to extreme metabolic disorders. The main manifestations are metabolic acidosis, phosphaturia, hypouricemia, glycosuria, osteopenia, systemic hyperaminoaciduria, proteinuria, and other extreme metabolic disorders. Currently, treatment is limited to supportive measures: replenishing lost solutes through fluid and electrolyte infusions, but chronic renal failure can still develop in adulthood. Therefore, early diagnosis may help in the treatment of the underlying defect. Once we have a better understanding of the pathophysiology of specific proximal tubule lesion subtypes, more targeted treatments for FRTS may emerge, and more targeted therapies may become available.

[0003] As early as 1986, Barker first proposed that the occurrence of cardiovascular and cerebrovascular diseases in adulthood originated from intrauterine growth retardation during fetal development. Subsequently, a large number of studies on the correlation between adverse environment during pregnancy, fetal birth weight and chronic diseases in adulthood have proposed a new concept of the origin of human diseases - Developmental Origins of Health and Disease (DOHaD). In recent years, research on the fetal developmental origin of diseases has been gradually shifted to the gamete development stage. Domestic and foreign researchers have found that adverse environmental factors during gamete formation and embryonic development in early life can affect offspring development, thereby significantly increasing the risk of offspring being susceptible to multiple diseases after birth, thus proposing the theory of "gamete-derived adult diseases" [2]. Studies have shown that adverse paternal environmental exposure before pregnancy (such as diet, exposure to exogenous substances, chronic stress, etc.) can lead to intrauterine growth retardation in offspring and susceptibility to multiple metabolic diseases after birth. For example, a father's high-fat, high-salt, and high-sugar diet can lead to chronic kidney disease in offspring, and paternal obesity can cause renal tubular damage in offspring. This suggests that adverse paternal environmental exposure before pregnancy is closely related to the long-term occurrence of diseases in offspring. Therefore, establishing a stable animal model of paternal proximal tubule dysfunction is of great significance for in-depth exploration of the pathogenesis of paternal proximal tubule dysfunction and analysis of paternal developmental origins. It also provides new ideas for guiding healthy paternal preconception, early warning and prevention.

[0004] For many years, reliable and reproducible experimental models have been very important in the study of human pathology, especially for multi-etiological diseases such as proximal tubule dysfunction. Currently developed animal models use transgenic knockout mice (such as megalin and cubilin) ​​or are induced by heavy metals [3]. Although some mechanisms leading to generalized proximal tubule dysfunction have been highlighted, they cannot reproduce all the features of proximal tubule dysfunction, and due to the important role of megalin and cubilin in development, no human diseases associated with the complete absence of these proteins have been described in humans, with only about 2% of animals surviving to adulthood. There are also reports of in vitro experiments on isolated rat renal tubules, but they can only briefly replicate the initial stage of renal dysfunction and are still of no value for subsequent testing of new treatments [4]. More and more studies suggest that proximal tubule dysfunction most often occurs in children, and the genetic causes are still unknown. Paternal proximal tubular dysfunction (PTD) is primarily caused by adverse pre-conception environmental exposure in the father, leading to PTD symptoms in offspring after birth. Its key characteristic is that the precipitating factor originates in utero, causing genetic programming alterations that persist into adulthood. Even without a specific triggering factor, PTD can still occur in adulthood. To date, no animal models of fetal PTD have been reported domestically or internationally. Therefore, constructing a simple, stable, and genetic animal model that simulates the pathophysiology of paternal PTD for research on its pathogenesis and prevention is a pressing issue in this field.

[0005] Caffeine is widely found in coffee, tea, chocolate, energy drinks, food supplements, and painkillers, and is one of the most widely used psychoactive drugs in the world. Survey data shows that coffee consumption in my country has increased by 15%–20% annually in recent years, and the average caffeine intake of men of reproductive age is 240 mg / day, about 1.5 times that of women. Therefore, caffeine exposure is more common in men before conception than in pregnant women. However, the impact of male caffeine intake during the preconception period on offspring development has not received widespread attention. Some evidence suggests that paternal health behaviors during the perinatal period may be related to offspring health outcomes; for example, paternal smoking or alcohol consumption during the perinatal period may lead to poorer offspring health (such as low birth weight, ADHD, and cognitive impairment). The inventors of this application found that paternal preconception caffeine exposure can construct a stable offspring IUGR model, and can also cause changes in sperm imprinting genes that are transmitted to offspring, leading to altered renal imprinting gene expression and proximal tubule dysfunction in offspring.

[0006] References:

[0007] 1.van der Wijst,J.,et al.,Learning Physiology From Inherited KidneyDisorders.Physiol Rev,2019.99(3):p.1575-1653.

[0008] 2.Fleming, TP, et al., Origins of lifetime health around the time ofconception: causes and consequences. Lancet, 2018.391(10132):p.1842-1852.

[0009] 3. Nielsen, R., EI Christensen, and H. Birn, Megalin and cubilin inproximal tubule protein reabsorption: from experimental models to human disease. Kidney Int, 2016.89(1):p.58-67.

[0010] 4. Gozalpour, E. and KSFenner, Current State of In vitro Cell-BasedRenal Models. Curr Drug Metab, 2018.19(4):p.310-326. Summary of the Invention

[0011] To address the shortcomings of existing animal models, this invention provides a method for constructing and applying an animal model of paternal proximal tubular dysfunction. This animal model has a high success rate, is effective and reliable, highly reproducible, simple and easy to implement, and can simulate the clinical characteristics of proximal tubular dysfunction, which is more consistent with the human genetic acquisition process.

[0012] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0013] In a first aspect, the present invention provides a method for constructing an animal model of paternal proximal renal tubule dysfunction, characterized in that:

[0014] S1: Select healthy male rodents (male rats) and administer 60 mg / kg of caffeine via intragastric gavage daily for eight weeks before mating and conception with female rats. Male rats are allowed free access to food.

[0015] S2: Male mice were injected with caffeine into their stomachs daily for 8 consecutive weeks and then mated with female mice. The pregnant mice gave birth naturally, resulting in the F1 generation. The birth date was taken as day 0 after birth. One day after birth, litters with 12 to 14 offspring were selected, and each litter was adjusted to have 6 male and 6 female offspring for nursing.

[0016] S3: The offspring (pups) are weaned 4 weeks after birth and the males and females are separated into different cages. They are then fed a normal diet until 12 weeks of age.

[0017] S4: Some offspring were raised until 32 weeks after birth, and proximal tubule transport and metabolism indicators were detected to comprehensively determine proximal tubule dysfunction, ultimately obtaining an animal model of paternal proximal tubule dysfunction.

[0018] As a preferred embodiment, in step S1, the male rodent is not limited to male rats and can be replaced with SPF-grade rodents including Wistar, SD rats, Kunming mice, or C57 mice.

[0019] Furthermore, in step S3, the normal diet is formulated to be the same as the formula for mouse and rat feed specified in the National Standard of the People's Republic of China GB14924.3-2001.

[0020] Furthermore, in step S4, the proximal tubule transport metabolism-related indicators are: blood and urine biochemical indicators, proximal tubule structure and transport protein and mRNA expression (including megalin, cublin, URAT1, GLUTs, OATs, OCTs, ABCs, etc.), proximal tubule-specific damage indicators (KIM-1, NAGL), and hematoxylin-eosin staining.

[0021] Secondly, the present invention provides an application of a paternal proximal renal tubule dysfunction animal model in screening for adverse environmental interferences or drugs from the father, characterized in that: the paternal proximal renal tubule dysfunction animal model is obtained by any of the above construction methods.

[0022] Thirdly, the present invention provides an application of a paternal proximal renal tubule dysfunction animal model in the early warning and intervention targets of paternal proximal renal tubule dysfunction, characterized in that: the paternal proximal renal tubule dysfunction animal model is obtained by any of the above construction methods.

[0023] Accordingly, the present invention also provides an application of a paternal proximal renal tubule dysfunction animal model in clinical guidance and monitoring improvement before conception, wherein the paternal proximal renal tubule dysfunction animal model is obtained by any of the above construction methods.

[0024] The technical principles and research process of this invention are as follows:

[0025] This invention investigates proximal tubule transport and metabolism indicators in offspring of fathers exposed to caffeine before pregnancy, confirming proximal tubule dysfunction in these offspring and establishing an animal model of paternal proximal tubule dysfunction. While some existing animal models can simulate proximal tubule dysfunction, they often exhibit significant genetic defects or single-drug effects, failing to mimic the developmental origins of the disorder and exhibiting substantial differences from the pathogenesis of human proximal tubule dysfunction. Caffeine, a common developmental toxin exposed daily, was used in this study. Fathers were exposed to caffeine before pregnancy for eight consecutive weeks, covering the entire spermatogenesis cycle, before mating with female mice to produce offspring. This animal model emphasizes the indirect effects on offspring, more closely mirroring the developmental changes in human fetal diseases from intrauterine to postnatal stages. It simulates the disease phenotype of paternal proximal tubule dysfunction, making it significant for studying the developmental mechanisms of paternal proximal tubule dysfunction and identifying early warning and intervention targets.

[0026] The advantages and beneficial effects of this invention are as follows:

[0027] 1. The modeling method of this invention is simple and reliable. Spermatogenesis in mammals consists of three continuous physiological processes: proliferation and division of spermatogonial stem cells, meiosis of spermatocytes, and formation of spermatocytes from haploid cells. In male rats, this process takes an average of 52-54 days. After completing the entire spermatogenesis cycle, the germ cells of male rats transform from undifferentiated stem cells into mature sperm with motility and fertilization capacity. Therefore, based on the above-mentioned dosage conversion criteria and spermatogenesis characteristics, this invention establishes a paternal pre-pregnancy caffeine exposure rat model by administering 60 mg / kg·d caffeine via gavage to Wistar rats for 8 weeks. Continuous administration for 8 weeks covers the entire spermatogenesis cycle of the rats, providing a reliable method for studying paternal proximal tubular dysfunction of the kidneys.

[0028] 2. This invention provides, for the first time, an animal model of paternal proximal tubule dysfunction and a method for constructing it, which solves the technical problem of the lack of animal models of hereditary proximal tubule dysfunction in the field and provides a foundation for the study of the pathogenesis and intervention strategies of fetal proximal tubule dysfunction.

[0029] 3. This invention uses an animal model to simulate the natural state of the human body by simulating the indirect effects of paternal exogenous exposure on offspring in order to study the pathogenesis and treatment, which is of particular significance for the study of preconception reproductive health.

[0030] 4. This invention allows for verification and observation of animal models from multiple perspectives, including genes, proteins, blood and urine metabolism, and pathology. It exhibits good reproducibility, and the experimental results are authentic, reliable, and convincing. In summary, this invention constructs an animal model of paternal proximal tubular dysfunction in offspring through pre-pregnancy caffeine exposure in the father. This model exhibits characteristic symptoms of proximal tubular dysfunction, such as blood and urine metabolic disorders, decreased proximal tubular structure, reduced transport proteins and mRNA, and pathological damage. These symptoms and pathological processes are consistent with those of human proximal tubular dysfunction and can reflect the severity of the disease. The model is highly controllable and stable. It can be used for pre-conception clinical guidance, research on the pathogenesis and prevention of paternal proximal tubular dysfunction, and for the development and research of new drugs. Attached Figure Description

[0031] Figure 1 This is a flowchart illustrating the experimental process for constructing and validating the paternal proximal tubule dysfunction animal model described in this invention.

[0032] Figure 1 In the study: A: Parental treatment with PPCE; B: Offspring acquisition; C: Collection of blood and urine biochemistry using metabolic cages; D: Detection of proximal tubule structure and transport parameters; E: Proximal tubule pathology in offspring.

[0033] Figure 2 This invention relates to the alteration of proximal tubule structure genes in offspring resulting from paternal caffeine exposure.

[0034] Figure 2 In the study: A: mRNA expression of AQP1, Megalin, Cubilin, and OAT1, ion transporter genes in the proximal tubules of F1 offspring during intrauterine use; B: mRNA expression of AQP1, Megalin, Cubilin, and OAT1, ion transporter genes in the proximal tubules of F1 offspring at 12 weeks of age; C: mRNA expression of AQP1, Megalin, Cubilin, and OAT1, ion transporter genes in the proximal tubules of F1 offspring at 32 weeks of age. * indicates P < 0.05 compared to the control group; ** indicates P < 0.01 compared to the control group.

[0035] Figure 3 This invention relates to changes in proximal tubule structure and specific damage indicators in offspring resulting from paternal caffeine exposure.

[0036] Figure 3 In the table: A: Hematoxylin-eosin staining; B: KIM-1 mRNA expression of proximal tubule-specific damage gene in F1 offspring at 12 and 32 weeks of age; C: NAGL mRNA expression of proximal tubule-specific damage gene in F1 offspring at 12 and 32 weeks of age. * indicates P < 0.05 compared with the control group; ** indicates P < 0.01 compared with the control group.

[0037] Figure 4 Key gene alterations in proximal tubule transporters of offspring exposed to paternal caffeine in the present invention

[0038] Figure 4 Among them: A: mRNA expressions of GLUT2, KCNQ1, CLDN2 and URAT1, ion transporter genes in the proximal tubules of F1 offspring at GD20 in utero; B: mRNA expressions of GLUT2, KCNQ1, CLDN2 and URAT1, ion transporter genes in the proximal tubules of F1 offspring at 12 weeks of age; C: mRNA expressions of GLUT2, KCNQ1, CLDN2 and URAT1, ion transporter genes in the proximal tubules of F1 offspring at 32 weeks of age. * indicates P < 0.05 compared with the control group; ** indicates P < 0.01 compared with the control group.

[0039] Figure 5 Key gene alterations in fatty acid oxidation of offspring exposed to paternal caffeine in the present invention

[0040] Figure 5 Among them: A: Cpt1a mRNA expressions in F1 offspring at 12 weeks, 20 weeks and 32 weeks of age; B: Acadm mRNA expressions in F1 offspring at 12 weeks, 20 weeks and 32 weeks of age; C: Acox mRNA expressions in F1 offspring at 12 weeks, 20 weeks and 32 weeks of age. * indicates P < 0.05 compared with the control group; ** indicates P < 0.01 compared with the control group. Detailed implementation manners

[0041] The following further elaborates on the technical content of the present invention in combination with specific embodiments and drawings.

[0042] Example 1: Construction of an animal model of paternal renal proximal tubule dysfunction

[0043] 1. Experimental animals:

[0044] SPF-grade healthy Wistar rats were purchased from Hubei Provincial Center for Disease Control and Prevention, and the animal license number is: SCXK(E)2020 0018. This invention has been approved by the Ethics Committee of the Medical School of Wuhan University and is strictly implemented in accordance with the relevant treatment guidelines of international experimental animal protection certification and evaluation institutions.

[0045] The experimental animals were housed in a barrier environment at a temperature of 22 - 25°C, a humidity of 50%, and a 12-hour day-night cycle.

[0046] 2. Experimental methods:

[0047] As Figure 1As shown, a paternal caffeine exposure rat model was established by administering caffeine via gavage for 8 weeks (covering the entire spermatogenesis cycle) to male rats (60 mg / kg / day, equivalent to a human exposure of 632 mg / day). After 8 weeks of continuous administration, the rats were mated with normal female rats at 16 weeks of age. Female and male rats were mated together at a 2:1 ratio at 7 PM daily. Pregnancy was confirmed the following morning by observing vaginal smears and observing viable sperm cells under a microscope, which was recorded as gestational day 0 (GD0).

[0048] Twenty male Wistar rats (weighing 260-300g) and forty female Wistar rats (weighing 200-240g) were used. After 7 days of free access to water and food, the rats were grouped together at a male:female ratio of 1:2. Vaginal smears were taken the following morning to identify pregnant rats, which were recorded as day 0 of gestation.

[0049] Some pregnant female rats were euthanized under GD20 anesthesia, and fetal tissue was collected, weighed, and collected for testing. Other pregnant rats gave birth naturally, and pups with litters of 12-14 were selected at postnatal week 1 (PW1). The litter size was then adjusted to 12 pups, ensuring an equal number of male and female pups. The pups were then raised normally until PW12 and PW32. Blood and urine samples were collected from all offspring rats for testing. Two days after this, the animals were euthanized under anesthesia, and serum and kidney samples were collected. The left kidney was cryopreserved at 80°C for protein and RT qPCR gene detection, and the right kidney was fixed in neutral formaldehyde, embedded, sectioned, and stained with H&E and immunohistochemical staining.

[0050] 3. Detection indicators and methods:

[0051] 3.1 Serum biochemical indicators

[0052] The kit was used to detect serum creatinine, blood urea nitrogen, serum sodium, serum potassium, serum uric acid, serum calcium, and blood glucose. Before the experiment, serum samples were thawed on ice and thoroughly mixed, and the kit was brought to room temperature. The absorbance of each tube was measured according to the kit instructions, with distilled water as a blank control. The concentrations of each biochemical indicator in the serum were calculated based on the standards.

[0053] 3.2 Urine biochemical indicators

[0054] Before euthanizing mice, 24-hour urine samples were collected from metabolic cages to measure urine volume, microalbumin, phosphorus, potassium, calcium, sodium, uric acid, glucose, cystatin C, and KIM-1. Urine samples were thawed on ice and thoroughly mixed before the experiment, and the kits were brought to room temperature. The absorbance of each tube was measured according to the kit instructions, using distilled water as a blank control. The concentrations of each biochemical indicator in the urine were calculated based on the standards.

[0055] 3.3 Observation of tissue morphology using hematoxylin-eosin staining:

[0056] After soaking the tissue in 10% formalin for 24 hours, it was dehydrated, embedded in paraffin, sectioned, and dewaxed to water. The sections were then immersed in hematoxylin staining solution for 5 minutes, rinsed with tap water for 15 minutes, differentiated with 1% hydrochloric acid and ethanol, bluing with ammonia and rinsing with running water, eosin for 3 minutes, dehydrated with a series of ethanol solutions, and cleared with xylene for 3-5 minutes. After mounting with neutral resin, the pathological changes of the proximal tubules were observed under an optical microscope.

[0057] 3.4 Design and preparation of specific primers for proximal tubule structure and key transport genes:

[0058] The complete template DNA sequence of the relevant target gene was retrieved using the Entrez Nucleotides database search system of NCBI. The template DNA sequence of the aforementioned gene was then added to the primer design software Primer Premier 5.0 to obtain primer sequences. Furthermore, the BLAST database was used to compare the designed primer sequences to obtain specific primer sequences. Primer information used in this section is shown in Table 1. In addition, the specificity of newly designed and synthesized primers needs to be tested before their first use. The melting curve of the PCR product was observed using an ABI StepOne Plus real-time quantitative PCR instrument. The specificity of the primers and the presence of primer dimers were determined based on the elution time and peak area of ​​the PCR product in the melting curve.

[0059] Table 1 Primer sequences

[0060]

[0061]

[0062] 3.5 Statistical Analysis

[0063] All results obtained in this invention represent at least three independent experiments. Experimental data were plotted and statistically analyzed using GraphPadPrism 8.0 software and expressed as mean ± standard error (Mean ± SEM). The Student's-Test paired test was used to determine statistical significance between the two groups; multiple group comparisons were performed using one-way ANOVA and Tukey's test. P < 0.05 was considered statistically significant.

[0064] 4. Experimental Results:

[0065] 4.1 Changes in blood and urine biochemical indicators

[0066] The changes in blood and urine biochemical parameters in adult offspring rats are shown in Tables 1, 2, and 3. At 16 weeks of age, compared with the control group, paternal caffeine exposure significantly increased 24-hour urinary glucose (P<0.05, Table 2) and urinary uric acid (P<0.01, Table 2) in offspring rats. At 20 weeks of age, compared with the control group, paternal caffeine exposure showed no change in serum creatinine and blood urea nitrogen, but significantly decreased serum potassium and calcium (P<0.05, Table 3), while serum chloride and sodium showed no significant changes. Compared with the control group, paternal caffeine exposure significantly increased 24-hour urinary potassium, calcium, and glucose excretion (P<0.05, Table 4), while urinary sodium and chloride showed no significant changes. The experimental results indicate that, compared with the control group, paternal caffeine exposure resulted in proximal renal tubular dysfunction in adult offspring rats.

[0067] Table 2: Changes in urinary biochemical parameters of F1 offspring at 16 weeks of age (mean±SEM, n=10)

[0068]

[0069] Table 3: Changes in blood biochemical parameters of F1 offspring at 20 weeks of age (mean±SEM, n=10)

[0070]

[0071] Table 4: Changes in urinary biochemical parameters of F1 offspring at 20 weeks of age (mean±SEM, n=10)

[0072]

[0073] 4.2 Changes in proximal renal tubule structure and specific injury-related indicators

[0074] The results of gene alterations in proximal tubule structure in offspring rats during intrauterine period, at 12 weeks of age, and at 32 weeks of age are as follows: Figure 2 As shown, compared with the control group, the expression of key genes for proximal tubule structure (megalin, cubilin, AQP1, OAT1) in offspring with paternal caffeine exposure was significantly decreased at all three time points. Figure 2 (AC). Meanwhile, such as Figure 3 As shown, paternal caffeine exposure resulted in a decreased endothelial-medullary ratio in offspring. Morphologically, at 12 and 32 weeks of age, proximal tubular epithelial cell shedding was observed, with hyaline, granular, or cellular casts visible within the lumen. Figure 3 In the middle A), proximal tubule-specific damage markers (KIM-1, NAGL) were significantly elevated. Figure 3 (Middle BC). The above results suggest that paternal caffeine exposure causes proximal tubule damage in offspring rats, which can persist into adulthood.

[0075] 4.3 Changes in proximal tubule-related transporter markers in F1 offspring rats after paternal caffeine exposure

[0076] like Figure 4 As shown, compared with the control group, the levels of proximal tubule-associated ion transporters—uric acid transporter URAT1, glucose transporter GLUT2, calcium transporter CLDN2, and potassium transporter KCNQ1—in offspring exposed to caffeine from the father were significantly decreased during intrauterine contact, at 12 weeks of age, and at 32 weeks of age. Figure 4 (AC). The experimental results show that paternal caffeine exposure causes proximal tubule transport dysfunction in offspring rats, which can persist into adulthood.

[0077] Example 2: Application of a paternal proximal tubular dysfunction animal model in the study of the pathogenesis of fetal proximal tubular dysfunction.

[0078] 1. Laboratory animals:

[0079] The experimental animals were the same as in Example 1.

[0080] 2. Experimental methods:

[0081] The experimental animals were the same as in Example 1.

[0082] 3. Detection indicators and methods:

[0083] 3.1 Design and preparation of specific primers for proximal tubule structure and key transport genes:

[0084] Primer design and preparation were as described in Example 1. Primer information is shown in Table 2.

[0085] Table 5 Primer sequences

[0086]

[0087] 3.2 Statistical analysis as described in Example 1

[0088] 4. Experimental Results:

[0089] Using the paternal renal proximal tubule dysfunction animal model established by the method in Example 1, fatty acid oxidation-related genes (acadm, cpt1a, acox) were detected in offspring rats at 12, 20, and 32 weeks of age. Results are as follows: Figure 5 As shown, qPCR revealed that, compared to the control group, paternal caffeine exposure in adult offspring resulted in a significant decrease or a decreasing trend in fatty acid oxidation-related genes at all three time points. Figure 5 The results suggest that pre-pregnancy caffeine exposure in the father can cause proximal tubule dysfunction in offspring rats, and the underlying mechanism is related to abnormal fatty acid metabolism.

[0090] The modeling method of this invention involves treating the father with caffeine for eight consecutive weeks. After birth, the offspring are routinely raised until 32 weeks. The experimental group showed disordered hematuria biochemical indicators, a significant decrease in proximal tubule structure and key transport genes, and a significant increase in specific damage indicators. Adult offspring showed proximal tubule epithelial cell shedding, and clear, granular, or cellular casts were visible in the lumen, exhibiting typical features similar to human proximal tubule dysfunction. This animal model has a high success rate, is effective and reliable, highly reproducible, and simple to implement. Furthermore, all PPCE offspring showed clinical features of proximal tubule dysfunction, making it an effective method for establishing paternal proximal tubule dysfunction. This model simulates the disease phenotype of paternal proximal tubule dysfunction and can be used to further explore the pathogenesis of paternal proximal tubule dysfunction, which is of great significance for elucidating paternal developmental origins. It also provides new ideas for guiding healthy paternal preconception, early warning, and prevention.

Claims

1. A method for constructing an animal model of paternal proximal renal tubule dysfunction, characterized in that: Includes the following steps: S1: Select healthy male rodents and administer 60 mg / kg of caffeine via intragastric gavage daily for eight weeks before mating and conception with female mice. The male mice are allowed free access to food. S2: Male rodents were injected with caffeine into their stomachs daily for 8 weeks and then mated with female mice. The pregnant mice gave birth naturally, resulting in the F1 generation. The day of birth was taken as day 0 after birth. One day after birth, litters with 12 to 14 offspring were selected, and each litter was adjusted to have 6 male and 6 female offspring for nursing. S3: The offspring are weaned 4 weeks after birth and the males and females are separated into different cages. They are then fed a normal diet until 12 weeks of age. S4: Some offspring were raised until 32 weeks after birth, and proximal tubule transport and metabolism indicators were detected to comprehensively determine proximal tubule dysfunction, ultimately obtaining an animal model of paternal proximal tubule dysfunction. In step S1, the rodents are SPF grade, including Wistar, SD rats, Kunming mice, or C57 mice. In step S3, the normal diet is formulated in the same way as the formula feed for mice and rats specified in the National Standard of the People's Republic of China GB14924.3-2001. In step S4, the proximal tubule transport and metabolism related indicators are: blood and urine biochemical indicators, proximal tubule structure and transport protein and mRNA expression, including Megalin, CUBN, URAT1, GLUTs, and OATs; proximal tubule specific damage indicators, namely KIM-1 and NAGL, and hematoxylin-eosin staining.

2. The application of a paternal renal proximal tubule dysfunction animal model in screening for adverse environmental disturbances or drugs during reproductive years, characterized in that: The paternal proximal tubule dysfunction animal model was obtained by the construction method in claim 1.

3. The application of an animal model of paternal proximal renal tubule dysfunction in screening early warning and intervention targets for paternal proximal renal tubule dysfunction, characterized in that: The paternal proximal tubule dysfunction animal model was obtained by the construction method in claim 1.

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