Evaluation of kidney injury and metabolomic analysis in adulthood in a non-obese hyperglycemic mouse model after birth with low birthweight

Evaluation of kidney injury and metabolomic analysis in adulthood in a non-obese hyperglycemic mouse model after birth with low birthweight | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of kidney injury and metabolomic analysis in adulthood in a non-obese hyperglycemic mouse model after birth with low birthweight Shoichi Shimizu, Nobuhiko Nagano, Daichi Katayama, Kengo Matsuda, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5773108/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jul, 2025 Read the published version in BMC Nephrology → Version 1 posted 4 You are reading this latest preprint version Abstract Background Low birthweight infants have high risk of developing chronic kidney disease (CKD) in later in life, however, the pathogenesis of this disease remains unclear. This study aimed to investigate the underlying mechanism using a low birthweight-non-obese hyperglycemic adulthood mouse model. Methods Pregnant ICR-strain mice underwent uterine artery ligation at day 16.5 of gestation to induce fetal hypoxia (ischemic group, I). Female newborns were weaned at 4 weeks of age and fed a normal diet until 8 weeks of age (n = 10). The group I was compared to the control group (C) regarding the body weight, tubular injury markers, renal function, pathology, and metabolome analysis. Results Group I were born with a low birth weight (group I: C = 1.4:1.9 g, p < 0.01), which persisted after birth. By 8 weeks of age, there were minimal changes in kidney histopathology between the two groups. However, group I showed an increase in markers for detection of CKD, such as urinary β2-microglobulin levels (group I༚C = 116:26 µg/L), albumin levels (group I༚C=0.14:0.07 mg/gCr) (both p < 0.01) and serum creatinine levels (group I༚C༝0.18:0.12 mg/dL, p < 0.05). Furthermore, kidney metabolomic analysis revealed notable differences between the two groups, particularly in succinic acid, S-adenosylmethionine, and N1-methyl-4-pyridone-5-carboxamide (4PY), which are closely linked to kidney injury. Conclusion The low birthweight-non-obese hyperglycemic mouse model may develop CKD in adulthood, potentially caused by increased renin activity related to succinic acid and tissue injury related to S-adenosylmethionine and 4PY. Chronic kidney disease (CKD) non-obese-hyperglycemia uterine artery ischemia metabolomic analysis developmental origins of health and disease (DOHaD) small-for-gestational-age (SGA) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction In Japan, although the birthrate is declining, the proportion of low birth weight infants has not decreased. Low birth weight infants, especially small-for-gestational-age (SGA) infants, are prone to developing chronic kidney disease (CKD) in the future [1], with several mechanisms known to cause this. Among these mechanisms, a decreased number of nephrons at birth leads to hyperfiltration of the residual glomeruli. Subsequently, elevated intraglomerular pressure can cause focal glomerulosclerosis, which results in hypertension, and ultimately, kidney injury, creating a negative loop [2]. It has also been hypothesized that the pathological mechanism of CKD involves metabolic abnormalities in the inner mitochondrial membrane via a breakdown in redox reactions, which leads to mitochondrial injury. Moreover, an increase in the adenosine monophosphate (AMP)/adenosine triphosphate ratio in the kidney causes intracellular energy insufficiency in AMP-activated protein kinase [3]. In experiments using fetal malnourished rats, we proposed that epigenetic abnormalities in stem cells in the kidney may lead to kidney injury and hypertension in adulthood [4]. However, this fetal malnutrition model may cause changes in the metabolic state and body environment of pregnant mothers due to an extremely low-protein diet, and it remains unclear whether this model can be applied to low birthweight infants in humans. Therefore, we successfully generated an SGA mouse model using intrauterine ischemia manipulation. We reported that this model develops hyperglycemia even after birth, despite being low weight, i.e., non-obese, and becoming an adult (a non-obese hyperglycemic mouse model that develops after birth with low birthweight, low birthweight-non-obese hyperglycemic mouse model) [5]. We sought to analyze these low birthweight-non-obese hyperglycemic model mice given that this model is thought to be closer to the actual clinical situation of low birthweight infants in humans. The purpose of this study was to clarify the presence of CKD in this mouse model by assessing kidney histomorphology and kidney function using urinalysis, blood analysis and pathological examination. We also performed metabolomic analysis of kidney tissue to elucidate the pathogenesis of kidney injury. The results may help to elucidate the mechanisms involved in CKD in low birthweight infants in adulthood. 2. Materials and methods 2.1 Creation of a low birthweight, non-obese, hyperglycemic mouse model This study was conducted in accordance with the ARRIVE guidelines, and the protocol was approved by the Nihon University Animal Care and Use Committee (protocol number: AP20MED003-1 [April 3, 2020]). ICR-strain mice (Sankyo Lab Service Co., Ltd. Tokyo, Japan) were obtained on gestational day 12. All mice were fed a normal diet (moisture: 7.9%, crude fat: 5.1%, crude protein: 23.1%, crude ash: 5.8%, crude fiber: 2.8%, and soluble solids: 55.3%; Oriental Yeast Co., Ltd., Tokyo, Japan) and had free access to water. Female mice were given the same diet and water until weaning after birth. On day 16.5 of pregnancy, the lower abdomen was incised under isoflurane inhalation anesthesia (induction 5%, maintenance 2%). In the ischemia group (group I), the mother mice were preheated to 37.5°C on a hot plate, the uterine artery was exposed, and the blood flow to the artery was blocked with a clip for 15 min to induce fetal hypoxia and malnutrition [6]. The uterine artery clip was removed, the fetus was returned to the mother’s abdomen, and the abdomen was sutured. The control group (group C) underwent a lower abdominal incision under similar anesthesia. The newborns were kept under the care of their mothers. Female newborns in both groups were weaned at 4 weeks of age and fed a normal diet until 8 weeks of age (Online Resource 1). The pups were weighed at birth and twice a week thereafter until they reached 8 weeks of age, at which point the weight gain plateaued. Mice aged 8 weeks are considered equivalent to adult humans [7]. 2.2 Confirmation of the kidney tissue injury phenotype Female newborns in groups I and C were weaned at 4 weeks of age and fed a normal diet until 8 weeks of age (n = 10 per group). The mice were weighed at birth and weekly thereafter until 8 weeks of age. At 7–8 weeks of age, the urinary β2-microglobulin and albumin levels were measured. For the measurements, 24-h urine samples were collected from mice housed in metabolic cages for experimental animals. The samples were stored at − 20°C, and urinary β2-microglobulin was measured by latex agglutination and albumin by immunoturbidimetry (SRL, Inc., Tokyo, Japan). Blood was then drawn from the heart, and the kidneys were removed at 8 weeks of age. Blood was centrifuged at 3000 rpm for 5 min at room temperature, and serum was stored at − 20°C. Serum creatinine levels were measured using a Dri-Chem slide (Fujifilm Corporation, Tokyo, Japan). The kidneys were fixed in 10% formalin followed by 70% alcohol replacement, and kidney sections were prepared. Periodic acid-Schiff staining was performed to assess the histological features. Glomerular counts, glomerular length diameters, and percentage of sclerotic glomerular were examined in all section prepared from each kidney. The glomerular injury score (GIS) was calculated based on the method outlined by Raij et al. [8] to determine the proportion of sclerotic glomeruli. 2.3 Metabolomic analysis Metabolomic analysis was performed to investigate kidney function and glucose metabolism. The metabolites were extracted as follows: Approximately 50 mg of frozen kidney tissue was removed from female mice (8 weeks old, n = 3 per group) and placed in homogenization tubes with zirconia beads (5 mmφ and 3 mmφ); next, an internal standard (H3304-1002, Human Metabolome Technologies, Inc. (HMT), Tsuruoka, Yamagata, Japan) was added, and the tissue was homogenized for 120 s at 4°C for two cycles at 1500 rpm using a bead shaker (Shake Master NEO, Bio Medical Science, Tokyo, Japan); the homogenate was centrifuged at 2300 x g for 5 min at 4°C, and the supernatant (400 µL) was centrifuged at 9100 × g for 120 min at 4°C using a Millipore 5-kDa cutoff filter (Human Metabolome Technologies, Inc.) to remove the polymers; and finally, the filtrate was dried under vacuum and redissolved in 50 µL Milli-Q water for metabolomic analysis. Metabolomic analysis was performed using capillary electrophoresis time-of-flight mass spectrometry as previously reported [9,10] using an Agilent CE capillary electrophoresis system (Agilent Technologies, Inc., Santa Clara, CA, USA). The spectrometer was scanned from 50 to 1000 m/z, and peaks were extracted using integrated software (Keio University, Tsuruoka, Yamagata, Japan) to obtain data on m/z, migration time, and peak area [11]. Peaks were determined using metabolite databases based on m/z values and migration times. Peak areas were standardized using internal standards and sample volumes to obtain relative metabolite levels. Principal component analysis and hierarchical cluster analysis were performed as previously reported [12]. The detected metabolites were plotted on a metabolic pathway map, as previously reported [13]. 2.4 Statistical analysis Statistical analysis was performed using JMP ver. 14. The Wilcoxon/Kruskal–Wallis test and Welch’s t-test were calculated, where statistical significance was set to p-value < 0.05. 3. Results 3.1 Birth weight and weight change up to 8 weeks of age The mean birth weight was 1.5 g in group I and 1.8 g in group C, and low birthweight pups were born due to intrauterine ischemia (p < 0.01) (Fig. 1 a). Group I had consistently underweight than group C thereafter, until at 8 weeks of age. (p < 0.01) (Fig. 1 b). 3.2 Urinary β2-microglobulin and albumin levels at 7 weeks of age The mean urinary β2-microglobulin (Fig. 2 a) and albumin (Fig. 2 b) levels at 7 weeks of age were significantly higher in group I than in group C (both p < 0.01). 3.3 Serum creatinine level at 8 weeks of age The mean serum creatinine level at 8 weeks was significantly higher in group I than in group C (p < 0.05) (Fig. 3 ). 3.4 Kidney pathology at 8 weeks of age The kidney pathology findings at 8 weeks of age were compared between groups, revealing no significant differences in glomerular counts or GIS (p = 0.75, p = 0.35). The glomerular length diameter was significantly longer in group I than in group C (p < 0.05) (Fig. 4 ). 3.5 Metabolome analysis Metabolomic analysis of the kidney revealed a clear difference between groups I and C in the heatmap display of principal component analysis and hierarchical cluster analysis(Fig. 5 ). Significant differences between the two groups were observed for the compounds outlined in Table 1 . Furthermore, when an investigation was performed based on the Human Metabolome Database, the compounds listed in Table 2 were classified as renal disease and uremic toxins; of these, the two groups showed a N1-methyl-4-pyridone-5-carboxamide (4PY) was significantly higher in group I than in group C. Table 1 Compounds for which a significant difference was observed between the two groups Comparative Analysis Group I a vs. C b Compound name Ratio p -value Argininosuccinic acid 1.5 0.021 Ascorbic acid 0.5 0.004 Cytidylic acid 0.7 0.032 Flavin adenine dinucleotide divalent 0.8 0.046 Hypotaurine 1.3 0.024 Inosine 0.8 0.034 Isovalerylcarnitine 2.4 0.012 myo -Inositol 1-phosphate myo -Inositol 3-phosphate 1.4 0.039 N1-Methyl-4-pyridone-5-carboxamide (4PY) 2.6 0.049 N 5 -Ethylglutamine 2.1 0.011 Ribulose 5-phosphate 0.7 0.008 S -Adenosylmethionine 0.5 0.002 S -Carboxymethylcysteine 1.6 0.029 Spermidine 1.5 0.024 Stachydrine 2 0.043 Succinic acid 1.8 0.005 Taurocholic acid 0.6 0.027 a: Ischemia group, b: Control group Table 2 Examination based on the Human Metabolome Database Comparative Analysis Group I a vs. C b Category Compound name Ratio p -value Renal failure, kidney disease, uremic toxin 3-Indoxylsulfuric acid 2.3 0.231 Renal failure Dimethylarginine 1.2 0.391 Renal failure, kidney stone Citric acid 1.4 0.476 Kidney disease, renal function Creatinine 1.7 0.127 Renal failure, aciduria, kidney stone Glyceric acid 2.3 0.085 Kidney disease, uremic toxin Guanidinosuccinic acid 1.4 0.255 Chronic renal failure His 1 0.913 Kidney disease Lys 1.2 0.344 Renal failure N -Acetylneuraminic acid 1 0.76 Renal failure N 1 -Methyl-4-pyridone-5-carboxamide (4PY) 2.6 0.049 Renal disease Symmetric dimethylarginine 1.3 0.295 Kidney failure Taurine 0.9 0.504 Chronic renal failure, hemodialysis Taurocyamine 1.1 0.579 Kidney failure Trimethylamine N -oxide 2.5 0.142 a: Ischemia group, b: Control group 4. Discussion A low birthweight-non-obese hyperglycemic mouse model was generated using intrauterine ischemia. The body weight of mice was monitored from birth, and blood and urine tests related to kidney function were performed in early adulthood. Metabolomic analysis and a histopathological search of kidney tissue were also performed. In a previously reported fetal growth restriction rat model, the histological findings at 32 weeks of age showed severe glomerular sclerosis and increased glomerular diameter due to long-term kidney dysfunction [14]. In contrast, in this experiment, the low birthweight-non-obese hyperglycemic model mice showed only slight histopathological changes at the relatively young age of 8 weeks. Pathological findings did not lead to the conclusion that the low birthweight-non-obese hyperglycemic model mice developed CKD. However, the low birthweight-non-obese hyperglycemic model mice were already showing signs of CKD, with elevated tubular injury markers and increased albuminuria. This result showed a similar tendency in a previously reported fetal growth restriction model rat that was examined at a younger age [15]. We also examined compounds that showed significant differences between the two groups in metabolomic analysis of the kidneys. Succinic acid, S-adenosylmethionine, and 4PY, which are known to be associated with kidney disease, were examined. The succinic acid content was significantly higher in group I than in group C. Hyperglycemia causes high succinate production in mitochondria. Previous reports have shown that succinate activates prorenin in the distal tubules where succinate receptors are present, producing renin and contributing to the onset of kidney injury [16], suggesting that the increase in renin activity induced by succinate may be involved in the onset of CKD in low birthweight-non-obese hyperglycemic model mice. S-adenosylmethionine, which showed significantly lower values in group I than in group C, is a direct methyl group donor of methionine [17]. Therefore, it is suggested that in the low birthweight-non-obese hyperglycemic mouse model, a shortage of S-adenosylmethionine in the tissues led to a decrease in the rate of methylation, which may have led to a decrease in the tissue repair ability of the kidney tissue. Physical property classification using the Human Metabolome Database revealed that 4PY, an indicator of kidney dysfunction and the final product of nicotinamide adenine dinucleotide (NAD), was significantly higher in group I than in group C. Poly (ADP-ribose) polymerase (PARP) plays an important role in the degradation of NAD. PARP is a nuclear enzyme that is deeply involved in various physiologically important events such as gene expression regulation, cell differentiation, apoptosis, DNA replication, and DNA repair [18]. It has been reported that DNA damage increases PARP activity several-fold [19]. As a result, intracellular NAD is rapidly depleted, resulting in the accumulation of nicotinamide. Nicotinamide is converted back to NAD or metabolized to 4PY [20]. In other words, 4PY increases at sites of DNA damage and can serve as a marker of tissue injury, including kidney tissue injury. Considering these mechanisms, the results of this metabolomic analysis showing that 4PY in kidney tissue was significantly higher in group I than in group C suggest that DNA damage may have occurred in kidney tissue in the low birthweight-non-obese hyperglycemic model mice. Furthermore, in a previous report using the same mouse model, we reported that serum NAD levels were significantly lower in group I than in group C (p = 0.010), which is in agreement with the 4PY movement in kidney tissue in this study [2]. In the same study, the reason for the change in NAD was proposed to be due to ischemia and reperfusion during the fetal period causing oxidative stress and a decrease in mitochondrial function [21–24]. This further reinforces the possibility that oxidative stress due to intrauterine ischemia in a low birthweight-non-obese hyperglycemic mouse model may cause mitochondrial dysfunction after birth and ultimately DNA damage in kidney tissue. Consequently, it is inferred that when the repair of DNA damage collapsed, CKD develops. A limitation of this study was that it was performed before actual histological changes were added due to the short rearing period. However, epigenetic changes acquired during the perinatal period and immediately after birth are a predisposition to cardiometabolic risk factors, including lifelong kidney disease [25]. Therefore, it is important to evaluate kidney tissue before the addition of histological changes. Furthermore, we did not create knockout models for each compound extracted via metabolomic analysis in this study; thus, we cannot prove that the effects of each compound are independent, and we cannot deny the possibility that the effects are due to multiple factors. These are issues to be considered in the future. 5. Conclusion In the low birthweight-non-obese hyperglycemic mouse model, kidney tubular injury and microalbuminuria were observed from early adulthood, and kidney function also changed. The possibility of CKD through long-term rearing was recognized, and the pathology was suggested to involve increased renin activity due to succinic acid and tissue injury due to S-adenosylmethionine and 4PY. Declarations Funding This research was supported by the Nihon University Research Grant (2022), Nihon University School of Medicine Alumni Association's 60th anniversary fund research grant (2023), the Grants-in-Aid for Young Scientists (grant number: 19K20194, 22K15908, 22K15446, and 22K17839), Scientific Research (C) (grant number: 21K11582 and 23K07258) of JSPS KAKENHI, and Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics (2023) Author contributions Conceptualization, Shoichi Shimizu, Nobuhiko Nagano and Ichiro Morioka ; methodology, Shoichi Shimizu, Nobuhiko Nagano, Daichi Katayama and Kimitaka Nakazaki and Ichiro Morioka; formal analysis and investigation, Shoichi Shimizu, Nobuhiko Nagano, Daichi Katayama and Kimitaka Nakazaki, Wataru Tokunaga, Ryoji Aoki and Kazumasa Fuwa; data curation, Shoichi Shimizu, Nobuhiko Nagano and Ichiro Morioka; writing—original draft preparation, Shoichi Shimizu, Nobuhiko Nagano and Ichiro Morioka; writing—review and editing, Shoichi Shimizu, Nobuhiko Nagano, Daichi Katayama and Kimitaka Nakazaki, Wataru Tokunaga, Ryoji Aoki and Kazumasa Fuwa; visualization, Shoichi Shimizu, Nobuhiko Nagano and Ichiro Morioka; supervision, Ichiro Morioka.; funding acquisition, Shoichi Shimizu, Nobuhiko Nagano, Kazumawa Fuwa. and Ryoji Aoki. All authors have read and agreed to the published version of the manuscript. Ethics declarations Ethics approval and consent to participate This study was conducted in accordance with the ARRIVE guidelines, and the protocol was approved by the Nihon University Animal Care and Use Committee (protocol number: AP20MED003-1 [April 3, 2020]). Consent for publication Not applicable. Competing interests The authors declare no competing interests. Clinical trial number Not applicable. References White SL, Perkovic V, Cass A, et al. Is low birth weight an antecedent of CKD in later life? A systematic review of observational studies. Am J Kidney Dis. 2009; 54:248-6.1 Brenner BM, Lawler EV, Mackenzie HS. The hyperfiltration theory: a new theory of hyperfiltration. a paradigm shift in nephrology. Kidney Int. 1996; 49:1774-7. Kikuchi H, Sasaki E, Nomura N, et al. Failure to sense energy depletion may be a novel therapeutic target in chronic kidney disease. Kidney Int. 2019; 95:. 123-37. 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Long-term renal tubular damage in intrauterine growth-restricted rats. Pediatr Int. 2018; 60:565-8. Toma I, Kang JJ, Sipos A, et al. Succinate receptor GPR91 provides a direct link between high glucose levels and renin release in murine and rabbit kidney. J Clin Invest. 2008; 118:2526-34. Obata F, Kuranaga E, Tomioka K, et al. Necrosis-driven systemic immune response alters SAM metabolism through the FOXO-GNMT axis. Cell Rep. 2014; 7:821-33. Burkle A. Physiology and pathophysiology of poly(ADP-ribosyl)-ation. Bioessays. 2001; 23:795-806. D'Amours D, Desnoyers S, D'Silva I, et al. Poly(ADP-ribosylreactions in the regulation of nuclear functions. Biochem J. 1999; 342:249-68. Rutkowski B, Slominska E, Szolkiewicz M, et al. N-methyl-2-pyridone-5-carboxamide: a novel uremic toxin? Kidney Int. 2003; 84:S19-21. Granger D, Kvietys, PR. Reperfusion injury and reactive oxygen species. The evolution of a concept. Redox Biol. 2015; 6:524-51. Crabtree MJ, Hale AB, Channon KM. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5773108","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":401892161,"identity":"250ec3ce-f23d-40b3-b430-daa4dfa9dccb","order_by":0,"name":"Shoichi 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Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kazumasa","middleName":"","lastName":"Fuwa","suffix":""},{"id":401892169,"identity":"7490f676-6343-406b-9754-9558d38181be","order_by":8,"name":"Ichiro Morioka","email":"","orcid":"","institution":"Nihon University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ichiro","middleName":"","lastName":"Morioka","suffix":""}],"badges":[],"createdAt":"2025-01-06 10:57:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5773108/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5773108/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12882-025-04290-1","type":"published","date":"2025-07-02T15:58:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73867707,"identity":"db27033c-38bc-4ae3-92d4-4b0a66f305f0","added_by":"auto","created_at":"2025-01-15 12:00:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24368,"visible":true,"origin":"","legend":"\u003cp\u003eBirth weight and changes in body weight gain\u003c/p\u003e\n\u003cp\u003e(a) The birth weight was measured on the first postnatal day. \u003cbr\u003e\n(b) Changes in weight gain from birth to 8 weeks of age\u003c/p\u003e\n\u003cp\u003eIschemia group: I, Control group: C\u003c/p\u003e\n\u003cp\u003eData are presented as the mean ± standard error of the mean (n = 10 per group).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5773108/v1/41257e5f5ea969f02bb82cac.png"},{"id":73865977,"identity":"39c6c4ee-90ee-4770-a1a3-05f4dc35b993","added_by":"auto","created_at":"2025-01-15 11:52:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15840,"visible":true,"origin":"","legend":"\u003cp\u003eMarkers of urinary tubular dysfunction\u003c/p\u003e\n\u003cp\u003e(a) Urinary β2-microglobulin at 7 weeks of age.\u003c/p\u003e\n\u003cp\u003e※Group C contained three samples with sensitivities of 10 μg/L or less.\u003c/p\u003e\n\u003cp\u003e(b) Urinary albumin/creatinine ratio at 7 weeks of age.\u003c/p\u003e\n\u003cp\u003eIschemia group: I, Control group: C\u003c/p\u003e\n\u003cp\u003eData are presented as the mean ± standard error of the mean (n = 6 per group).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5773108/v1/1a4553bee503fbe6abb06030.png"},{"id":73865987,"identity":"ef4dc394-d6ac-4198-aceb-90af536d1808","added_by":"auto","created_at":"2025-01-15 11:52:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7658,"visible":true,"origin":"","legend":"\u003cp\u003eSerum creatinine level at 8 weeks of age\u003c/p\u003e\n\u003cp\u003eIschemia group: I, Control group: C\u003c/p\u003e\n\u003cp\u003eData are presented as the mean ± standard error of the mean (n = 6 per group).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5773108/v1/8350d8725574a2e7d0e76634.png"},{"id":73865986,"identity":"f5b5820b-2d46-427b-815a-1131ba891fc0","added_by":"auto","created_at":"2025-01-15 11:52:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":113198,"visible":true,"origin":"","legend":"\u003cp\u003ePathological findings of the kidney\u003c/p\u003e\n\u003cp\u003e(a) Pathological image. Periodic acid-Schiff stain (×200). (b) Number of glomeruli.\u003cbr\u003e\n(c) Glomerular long axis. (d) Glomerular injury score.\u003c/p\u003e\n\u003cp\u003eIschemia group: I, Control group: C\u003cbr\u003e\nData are presented as the mean ± standard error of the mean (n = 6 per group).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5773108/v1/8c0f2a89bf40d588ec7e5e72.png"},{"id":73867709,"identity":"bb03a9ef-4bd9-4a6d-a488-f2fb70d6b199","added_by":"auto","created_at":"2025-01-15 12:00:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":75489,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolite analyses of kidney tissue\u003c/p\u003e\n\u003cp\u003e(a) Principal component (PC) analysis.\u003c/p\u003e\n\u003cp\u003e(b) Heatmap display of the hierarchical cluster analysis.\u003c/p\u003e\n\u003cp\u003eIschemia group: I, Control group: C\u003c/p\u003e\n\u003cp\u003en = 3 per group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5773108/v1/f9f37deb944c21c0ab2d9d8d.png"},{"id":86179894,"identity":"009a8370-9caf-4bdb-a483-375b09ad12fe","added_by":"auto","created_at":"2025-07-07 16:20:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":898824,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5773108/v1/c7a24395-864b-46eb-b2af-90fbe3a3d43d.pdf"},{"id":73865980,"identity":"a01dbaf9-57b4-4ca6-898f-c3eced3e9cdf","added_by":"auto","created_at":"2025-01-15 11:52:36","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":203506,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5supplementary.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5773108/v1/d990100c6815405cef2f1ccd.xlsx"},{"id":73865979,"identity":"6f820773-eff8-4172-8ab7-b2a2701ddb7b","added_by":"auto","created_at":"2025-01-15 11:52:36","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":279170,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5773108/v1/ca46130e6a01d18f3577d741.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of kidney injury and metabolomic analysis in adulthood in a non-obese hyperglycemic mouse model after birth with low birthweight","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn Japan, although the birthrate is declining, the proportion of low birth weight infants has not decreased. Low birth weight infants, especially small-for-gestational-age (SGA) infants, are prone to developing chronic kidney disease (CKD) in the future [1], with several mechanisms known to cause this. Among these mechanisms, a decreased number of nephrons at birth leads to hyperfiltration of the residual glomeruli. Subsequently, elevated intraglomerular pressure can cause focal glomerulosclerosis, which results in hypertension, and ultimately, kidney injury, creating a negative loop [2]. It has also been hypothesized that the pathological mechanism of CKD involves metabolic abnormalities in the inner mitochondrial membrane via a breakdown in redox reactions, which leads to mitochondrial injury. Moreover, an increase in the adenosine monophosphate (AMP)/adenosine triphosphate ratio in the kidney causes intracellular energy insufficiency in AMP-activated protein kinase [3].\u003c/p\u003e \u003cp\u003eIn experiments using fetal malnourished rats, we proposed that epigenetic abnormalities in stem cells in the kidney may lead to kidney injury and hypertension in adulthood [4]. However, this fetal malnutrition model may cause changes in the metabolic state and body environment of pregnant mothers due to an extremely low-protein diet, and it remains unclear whether this model can be applied to low birthweight infants in humans. Therefore, we successfully generated an SGA mouse model using intrauterine ischemia manipulation. We reported that this model develops hyperglycemia even after birth, despite being low weight, i.e., non-obese, and becoming an adult (a non-obese hyperglycemic mouse model that develops after birth with low birthweight, low birthweight-non-obese hyperglycemic mouse model) [5]. We sought to analyze these low birthweight-non-obese hyperglycemic model mice given that this model is thought to be closer to the actual clinical situation of low birthweight infants in humans.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to clarify the presence of CKD in this mouse model by assessing kidney histomorphology and kidney function using urinalysis, blood analysis and pathological examination. We also performed metabolomic analysis of kidney tissue to elucidate the pathogenesis of kidney injury. The results may help to elucidate the mechanisms involved in CKD in low birthweight infants in adulthood.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e2.1 Creation of a low birthweight, non-obese, hyperglycemic mouse model\u003c/p\u003e \u003cp\u003e This study was conducted in accordance with the ARRIVE guidelines, and the protocol was approved by the Nihon University Animal Care and Use Committee (protocol number: AP20MED003-1 [April 3, 2020]). ICR-strain mice (Sankyo Lab Service Co., Ltd. Tokyo, Japan) were obtained on gestational day 12. All mice were fed a normal diet (moisture: 7.9%, crude fat: 5.1%, crude protein: 23.1%, crude ash: 5.8%, crude fiber: 2.8%, and soluble solids: 55.3%; Oriental Yeast Co., Ltd., Tokyo, Japan) and had free access to water. Female mice were given the same diet and water until weaning after birth. On day 16.5 of pregnancy, the lower abdomen was incised under isoflurane inhalation anesthesia (induction 5%, maintenance 2%). In the ischemia group (group I), the mother mice were preheated to 37.5\u0026deg;C on a hot plate, the uterine artery was exposed, and the blood flow to the artery was blocked with a clip for 15 min to induce fetal hypoxia and malnutrition [6]. The uterine artery clip was removed, the fetus was returned to the mother\u0026rsquo;s abdomen, and the abdomen was sutured. The control group (group C) underwent a lower abdominal incision under similar anesthesia. The newborns were kept under the care of their mothers. Female newborns in both groups were weaned at 4 weeks of age and fed a normal diet until 8 weeks of age (Online Resource 1). The pups were weighed at birth and twice a week thereafter until they reached 8 weeks of age, at which point the weight gain plateaued. Mice aged 8 weeks are considered equivalent to adult humans [7].\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Confirmation of the kidney tissue injury phenotype\u003c/h2\u003e \u003cp\u003eFemale newborns in groups I and C were weaned at 4 weeks of age and fed a normal diet until 8 weeks of age (n\u0026thinsp;=\u0026thinsp;10 per group). The mice were weighed at birth and weekly thereafter until 8 weeks of age. At 7\u0026ndash;8 weeks of age, the urinary β2-microglobulin and albumin levels were measured. For the measurements, 24-h urine samples were collected from mice housed in metabolic cages for experimental animals. The samples were stored at \u0026minus;\u0026thinsp;20\u0026deg;C, and urinary β2-microglobulin was measured by latex agglutination and albumin by immunoturbidimetry (SRL, Inc., Tokyo, Japan). Blood was then drawn from the heart, and the kidneys were removed at 8 weeks of age.\u003c/p\u003e \u003cp\u003eBlood was centrifuged at 3000 rpm for 5 min at room temperature, and serum was stored at \u0026minus;\u0026thinsp;20\u0026deg;C. Serum creatinine levels were measured using a Dri-Chem slide (Fujifilm Corporation, Tokyo, Japan).\u003c/p\u003e \u003cp\u003eThe kidneys were fixed in 10% formalin followed by 70% alcohol replacement, and kidney sections were prepared. Periodic acid-Schiff staining was performed to assess the histological features. Glomerular counts, glomerular length diameters, and percentage of sclerotic glomerular were examined in all section prepared from each kidney. The glomerular injury score (GIS) was calculated based on the method outlined by Raij et al. [8] to determine the proportion of sclerotic glomeruli.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Metabolomic analysis\u003c/h2\u003e \u003cp\u003eMetabolomic analysis was performed to investigate kidney function and glucose metabolism. The metabolites were extracted as follows: Approximately 50 mg of frozen kidney tissue was removed from female mice (8 weeks old, n\u0026thinsp;=\u0026thinsp;3 per group) and placed in homogenization tubes with zirconia beads (5 mmφ and 3 mmφ); next, an internal standard (H3304-1002, Human Metabolome Technologies, Inc. (HMT), Tsuruoka, Yamagata, Japan) was added, and the tissue was homogenized for 120 s at 4\u0026deg;C for two cycles at 1500 rpm using a bead shaker (Shake Master NEO, Bio Medical Science, Tokyo, Japan); the homogenate was centrifuged at 2300 x g for 5 min at 4\u0026deg;C, and the supernatant (400 \u0026micro;L) was centrifuged at 9100 \u0026times; g for 120 min at 4\u0026deg;C using a Millipore 5-kDa cutoff filter (Human Metabolome Technologies, Inc.) to remove the polymers; and finally, the filtrate was dried under vacuum and redissolved in 50 \u0026micro;L Milli-Q water for metabolomic analysis. Metabolomic analysis was performed using capillary electrophoresis time-of-flight mass spectrometry as previously reported [9,10] using an Agilent CE capillary electrophoresis system (Agilent Technologies, Inc., Santa Clara, CA, USA). The spectrometer was scanned from 50 to 1000 m/z, and peaks were extracted using integrated software (Keio University, Tsuruoka, Yamagata, Japan) to obtain data on m/z, migration time, and peak area [11]. Peaks were determined using metabolite databases based on m/z values and migration times. Peak areas were standardized using internal standards and sample volumes to obtain relative metabolite levels. Principal component analysis and hierarchical cluster analysis were performed as previously reported [12]. The detected metabolites were plotted on a metabolic pathway map, as previously reported [13].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using JMP ver. 14. The Wilcoxon/Kruskal\u0026ndash;Wallis test and Welch\u0026rsquo;s t-test were calculated, where statistical significance was set to p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Birth weight and weight change up to 8 weeks of age\u003c/h2\u003e \u003cp\u003eThe mean birth weight was 1.5 g in group I and 1.8 g in group C, and low birthweight pups were born due to intrauterine ischemia (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Group I had consistently underweight than group C thereafter, until at 8 weeks of age. (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Urinary β2-microglobulin and albumin levels at 7 weeks of age\u003c/h2\u003e \u003cp\u003eThe mean urinary β2-microglobulin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and albumin (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) levels at 7 weeks of age were significantly higher in group I than in group C (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Serum creatinine level at 8 weeks of age\u003c/h2\u003e \u003cp\u003eThe mean serum creatinine level at 8 weeks was significantly higher in group I than in group C (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Kidney pathology at 8 weeks of age\u003c/h2\u003e \u003cp\u003eThe kidney pathology findings at 8 weeks of age were compared between groups, revealing no significant differences in glomerular counts or GIS (p\u0026thinsp;=\u0026thinsp;0.75, p\u0026thinsp;=\u0026thinsp;0.35). The glomerular length diameter was significantly longer in group I than in group C (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Metabolome analysis\u003c/h2\u003e \u003cp\u003eMetabolomic analysis of the kidney revealed a clear difference between groups I and C in the heatmap display of principal component analysis and hierarchical cluster analysis(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Significant differences between the two groups were observed for the compounds outlined in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Furthermore, when an investigation was performed based on the Human Metabolome Database, the compounds listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e were classified as renal disease and uremic toxins; of these, the two groups showed a N1-methyl-4-pyridone-5-carboxamide (4PY) was significantly higher in group I than in group C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCompounds for which a significant difference was observed between the two groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eComparative Analysis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGroup I\u003csup\u003ea\u003c/sup\u003e vs. C\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRatio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArgininosuccinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAscorbic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCytidylic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavin adenine dinucleotide divalent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypotaurine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.024\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.034\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsovalerylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003emyo\u003c/em\u003e-Inositol 1-phosphate \u003cem\u003emyo\u003c/em\u003e-Inositol 3-phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.039\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN1-Methyl-4-pyridone-5-carboxamide (4PY)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.049\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eN\u003c/em\u003e\u003csup\u003e5\u003c/sup\u003e-Ethylglutamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRibulose 5-phosphate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS\u003c/em\u003e-Adenosylmethionine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eS\u003c/em\u003e-Carboxymethylcysteine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.029\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpermidine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.024\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStachydrine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.043\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSuccinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTaurocholic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003ea: Ischemia group, b: Control group\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExamination based on the Human Metabolome Database\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eComparative Analysis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eGroup I\u003csup\u003ea\u003c/sup\u003e vs. C\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompound name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRatio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenal failure, kidney disease, uremic toxin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-Indoxylsulfuric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.231\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenal failure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDimethylarginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.391\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenal failure, kidney stone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCitric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.476\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney disease, renal function\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCreatinine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.127\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenal failure, aciduria, kidney stone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlyceric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.085\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney disease, uremic toxin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGuanidinosuccinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.255\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChronic renal failure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.913\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.344\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenal failure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eN\u003c/em\u003e-Acetylneuraminic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenal failure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eN\u003c/em\u003e\u003csup\u003e1\u003c/sup\u003e-Methyl-4-pyridone-5-carboxamide (4PY)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.049\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRenal disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSymmetric dimethylarginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.295\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney failure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTaurine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.504\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChronic renal failure, hemodialysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTaurocyamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.579\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney failure\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrimethylamine \u003cem\u003eN\u003c/em\u003e-oxide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.142\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003ea: Ischemia group, b: Control group\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eA low birthweight-non-obese hyperglycemic mouse model was generated using intrauterine ischemia. The body weight of mice was monitored from birth, and blood and urine tests related to kidney function were performed in early adulthood. Metabolomic analysis and a histopathological search of kidney tissue were also performed.\u003c/p\u003e \u003cp\u003eIn a previously reported fetal growth restriction rat model, the histological findings at 32 weeks of age showed severe glomerular sclerosis and increased glomerular diameter due to long-term kidney dysfunction [14]. In contrast, in this experiment, the low birthweight-non-obese hyperglycemic model mice showed only slight histopathological changes at the relatively young age of 8 weeks. Pathological findings did not lead to the conclusion that the low birthweight-non-obese hyperglycemic model mice developed CKD. However, the low birthweight-non-obese hyperglycemic model mice were already showing signs of CKD, with elevated tubular injury markers and increased albuminuria. This result showed a similar tendency in a previously reported fetal growth restriction model rat that was examined at a younger age [15].\u003c/p\u003e \u003cp\u003eWe also examined compounds that showed significant differences between the two groups in metabolomic analysis of the kidneys. Succinic acid, S-adenosylmethionine, and 4PY, which are known to be associated with kidney disease, were examined. The succinic acid content was significantly higher in group I than in group C. Hyperglycemia causes high succinate production in mitochondria. Previous reports have shown that succinate activates prorenin in the distal tubules where succinate receptors are present, producing renin and contributing to the onset of kidney injury [16], suggesting that the increase in renin activity induced by succinate may be involved in the onset of CKD in low birthweight-non-obese hyperglycemic model mice. S-adenosylmethionine, which showed significantly lower values in group I than in group C, is a direct methyl group donor of methionine [17]. Therefore, it is suggested that in the low birthweight-non-obese hyperglycemic mouse model, a shortage of S-adenosylmethionine in the tissues led to a decrease in the rate of methylation, which may have led to a decrease in the tissue repair ability of the kidney tissue.\u003c/p\u003e \u003cp\u003ePhysical property classification using the Human Metabolome Database revealed that 4PY, an indicator of kidney dysfunction and the final product of nicotinamide adenine dinucleotide (NAD), was significantly higher in group I than in group C.\u003c/p\u003e \u003cp\u003ePoly (ADP-ribose) polymerase (PARP) plays an important role in the degradation of NAD. PARP is a nuclear enzyme that is deeply involved in various physiologically important events such as gene expression regulation, cell differentiation, apoptosis, DNA replication, and DNA repair [18]. It has been reported that DNA damage increases PARP activity several-fold [19]. As a result, intracellular NAD is rapidly depleted, resulting in the accumulation of nicotinamide. Nicotinamide is converted back to NAD or metabolized to 4PY [20]. In other words, 4PY increases at sites of DNA damage and can serve as a marker of tissue injury, including kidney tissue injury. Considering these mechanisms, the results of this metabolomic analysis showing that 4PY in kidney tissue was significantly higher in group I than in group C suggest that DNA damage may have occurred in kidney tissue in the low birthweight-non-obese hyperglycemic model mice.\u003c/p\u003e \u003cp\u003eFurthermore, in a previous report using the same mouse model, we reported that serum NAD levels were significantly lower in group I than in group C (p\u0026thinsp;=\u0026thinsp;0.010), which is in agreement with the 4PY movement in kidney tissue in this study [2]. In the same study, the reason for the change in NAD was proposed to be due to ischemia and reperfusion during the fetal period causing oxidative stress and a decrease in mitochondrial function [21\u0026ndash;24]. This further reinforces the possibility that oxidative stress due to intrauterine ischemia in a low birthweight-non-obese hyperglycemic mouse model may cause mitochondrial dysfunction after birth and ultimately DNA damage in kidney tissue. Consequently, it is inferred that when the repair of DNA damage collapsed, CKD develops.\u003c/p\u003e \u003cp\u003eA limitation of this study was that it was performed before actual histological changes were added due to the short rearing period. However, epigenetic changes acquired during the perinatal period and immediately after birth are a predisposition to cardiometabolic risk factors, including lifelong kidney disease [25]. Therefore, it is important to evaluate kidney tissue before the addition of histological changes. Furthermore, we did not create knockout models for each compound extracted via metabolomic analysis in this study; thus, we cannot prove that the effects of each compound are independent, and we cannot deny the possibility that the effects are due to multiple factors. These are issues to be considered in the future.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn the low birthweight-non-obese hyperglycemic mouse model, kidney tubular injury and microalbuminuria were observed from early adulthood, and kidney function also changed. The possibility of CKD through long-term rearing was recognized, and the pathology was suggested to involve increased renin activity due to succinic acid and tissue injury due to S-adenosylmethionine and 4PY.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;This research was supported by the Nihon University Research Grant (2022), Nihon University School of Medicine Alumni Association's 60th anniversary fund research grant (2023), the Grants-in-Aid for Young Scientists (grant number: 19K20194, 22K15908, 22K15446, and 22K17839), Scientific Research (C) (grant number: 21K11582 and 23K07258) of JSPS KAKENHI, and Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics (2023)\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eConceptualization, Shoichi Shimizu, Nobuhiko Nagano and Ichiro Morioka ; methodology, Shoichi Shimizu, Nobuhiko Nagano, Daichi Katayama and Kimitaka Nakazaki and Ichiro Morioka; formal analysis and \u0026nbsp; investigation, Shoichi Shimizu, Nobuhiko Nagano, Daichi Katayama and Kimitaka Nakazaki, Wataru Tokunaga, Ryoji Aoki and Kazumasa Fuwa; data curation, Shoichi Shimizu, Nobuhiko Nagano and Ichiro Morioka; writing—original draft preparation, Shoichi Shimizu, Nobuhiko Nagano and Ichiro Morioka; writing—review and editing, Shoichi Shimizu, Nobuhiko Nagano, Daichi Katayama and Kimitaka Nakazaki, Wataru Tokunaga, Ryoji Aoki and Kazumasa Fuwa; visualization, Shoichi Shimizu, Nobuhiko Nagano and Ichiro Morioka; supervision, Ichiro Morioka.; funding acquisition, Shoichi Shimizu, Nobuhiko Nagano, Kazumawa Fuwa. and Ryoji Aoki. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eEthics declarations\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the ARRIVE guidelines, and the protocol was approved by the Nihon University Animal Care and Use Committee (protocol number: AP20MED003-1 [April 3, 2020]).\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eClinical trial number\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWhite SL, Perkovic V, Cass A, et al. Is low birth weight an antecedent of CKD in later life? A systematic review of observational studies. 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Capillary electrophoresis mass spectrometry-based saliva metabolomics identified oral, breast and pancreatic cancer-specific profiles. Metabolomics. 2009; 6:78\u0026ndash;95.\u003c/li\u003e\n\u003cli\u003eYamamoto H, Fujimori T, Sato H, et al. Statistical hypothesis testing of factor loading in principal component analysis and its application to metabolite set enrichment analysis. BMC Bioinform. 2014;15.\u003c/li\u003e\n\u003cli\u003eJunker BH, Klukas C, Schreiber F. VANTED: A system for advanced data analysis and visualization in the context of biological networks. BMC Bioinformatics. 2006; 7:109.\u003c/li\u003e\n\u003cli\u003eMurano Y, Nishizaki N, Endo A et al. Evaluation of kidney dysfunction and angiotensinogen as an early novel biomarker of intrauterine growth restricted offspring rats. Pediatr Res. 2015; 78:678-82.\u003c/li\u003e\n\u003cli\u003eMurano Y, Shoji H, Hara T, et al. Long-term renal tubular damage in intrauterine growth-restricted rats. 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Microcirculation. 2011; 18:304-11.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-nephrology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnep","sideBox":"Learn more about [BMC Nephrology](http://bmcnephrol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bnep/default.aspx","title":"BMC Nephrology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chronic kidney disease (CKD), non-obese-hyperglycemia, uterine artery ischemia, metabolomic analysis, developmental origins of health and disease (DOHaD), small-for-gestational-age (SGA)","lastPublishedDoi":"10.21203/rs.3.rs-5773108/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5773108/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eLow birthweight infants have high risk of developing chronic kidney disease (CKD) in later in life, however, the pathogenesis of this disease remains unclear. This study aimed to investigate the underlying mechanism using a low birthweight-non-obese hyperglycemic adulthood mouse model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePregnant ICR-strain mice underwent uterine artery ligation at day 16.5 of gestation to induce fetal hypoxia (ischemic group, I). Female newborns were weaned at 4 weeks of age and fed a normal diet until 8 weeks of age (n\u0026thinsp;=\u0026thinsp;10). The group I was compared to the control group (C) regarding the body weight, tubular injury markers, renal function, pathology, and metabolome analysis.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGroup I were born with a low birth weight (group I: C\u0026thinsp;=\u0026thinsp;1.4:1.9 g, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), which persisted after birth. By 8 weeks of age, there were minimal changes in kidney histopathology between the two groups. However, group I showed an increase in markers for detection of CKD, such as urinary β2-microglobulin levels (group I༚C\u0026thinsp;=\u0026thinsp;116:26 \u0026micro;g/L), albumin levels (group I༚C=0.14:0.07 mg/gCr) (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and serum creatinine levels (group I༚C༝0.18:0.12 mg/dL, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, kidney metabolomic analysis revealed notable differences between the two groups, particularly in succinic acid, S-adenosylmethionine, and N1-methyl-4-pyridone-5-carboxamide (4PY), which are closely linked to kidney injury.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe low birthweight-non-obese hyperglycemic mouse model may develop CKD in adulthood, potentially caused by increased renin activity related to succinic acid and tissue injury related to S-adenosylmethionine and 4PY.\u003c/p\u003e","manuscriptTitle":"Evaluation of kidney injury and metabolomic analysis in adulthood in a non-obese hyperglycemic mouse model after birth with low birthweight","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-15 11:52:31","doi":"10.21203/rs.3.rs-5773108/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-01-14T08:13:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-13T13:21:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-13T13:18:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Nephrology","date":"2025-01-06T10:37:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-nephrology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnep","sideBox":"Learn more about [BMC Nephrology](http://bmcnephrol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bnep/default.aspx","title":"BMC Nephrology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"08ddc5f5-4372-4e75-90a9-85649682fcdc","owner":[],"postedDate":"January 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:12:55+00:00","versionOfRecord":{"articleIdentity":"rs-5773108","link":"https://doi.org/10.1186/s12882-025-04290-1","journal":{"identity":"bmc-nephrology","isVorOnly":false,"title":"BMC Nephrology"},"publishedOn":"2025-07-02 15:58:30","publishedOnDateReadable":"July 2nd, 2025"},"versionCreatedAt":"2025-01-15 11:52:31","video":"","vorDoi":"10.1186/s12882-025-04290-1","vorDoiUrl":"https://doi.org/10.1186/s12882-025-04290-1","workflowStages":[]},"version":"v1","identity":"rs-5773108","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5773108","identity":"rs-5773108","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}