Irbesartan ameliorates kidney injury and anemia in a Col4a5-mutant mouse model of Alport syndrome with CKD pathology

Irbesartan ameliorates kidney injury and anemia in a Col4a5-mutant mouse model of Alport syndrome with CKD pathology | 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 Article Irbesartan ameliorates kidney injury and anemia in a Col4a5-mutant mouse model of Alport syndrome with CKD pathology Takumi Sugawara, Mitsuharu Matsumoto, Masami Yamamoto, Masashi Aoyama, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8786563/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Alport syndrome, caused by mutations in type IV collagen genes, leads to progressive proteinuria, anemia, and glomerulosclerosis, ultimately resulting in renal failure. It also shares pathological features with chronic kidney disease (C KD). Previous knockout models of Col4a3 or Col4a5 recapitulated renal disease but were less translatable to the human condition, which arises from specific genetic variants rather than complete gene loss. To address this limitation, we established a Col4a5 R471* mutant mouse carrying a clinically relevant mutation and evaluated its responsiveness to the angiotensin receptor blocker (ARB) irbesartan as a benchmark for translational utility in ameliorating kidney injury and anemia. Col4a5 R471* mutant mice exhibited significant increases in the urine albumin–creatinine ratio (UACR) and blood urea nitrogen, along with age-dependent decreases in glomerular filtration rate. In addition, changes in anemia-related factors—such as red blood cell count, hemoglobin level, and hematocrit value—were observed. Irbesartan treatment significantly reduced UACR and urinary neutrophil gelatinase–associated lipocalin levels and improved anemia-related factors and proinflammatory transcript expression. These results provide the first preclinical evidence that an ARB can improve kidney injury and anemia in Col4a5 R471* mutant mice, highlighting the model’s translational relevance for CKD drug evaluation. Health sciences/Diseases Biological sciences/Genetics Health sciences/Nephrology Alport syndrome angiotensin receptor blocker Irbesartan anemia collagen 4a5 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Alport syndrome (AS) is an inherited form of chronic kidney disease (CKD) characterized by progressive glomerular damage that ultimately leads to kidney failure and anemia. Its prevalence is estimated to range from 1 in 2,000 to 1 in 53,000 individuals, depending on diagnostic criteria and the extent of genetic screening. 1 – 3 Mutations in the type IV collagen genes— COL4A3 , COL4A4 , and COL4A5 —cause structural abnormalities of the glomerular basement membrane (GBM), which contribute to the pathogenesis of Alport syndrome. 4 – 7 These genes encode the α3, α4, and α5 chains of type IV collagen, which are integral components of the mature GBM. A large-scale whole-exome sequencing study revealed that mutations in COL4A3 , COL4A4 , and COL4A5 collectively account for nearly 30% of all monogenic CKD cases, establishing AS as the second most common hereditary nephropathy after autosomal dominant polycystic kidney disease. 8 This finding underscores the broader clinical relevance of AS, demonstrating that it is not only a rare disease but also a significant contributor to the overall burden of hereditary renal disorders. Approximately 80% of AS cases are X-linked (XLAS) and result from COL4A5 mutations, whereas the remaining cases exhibit autosomal recessive or autosomal dominant inheritance due to COL4A3 or COL4A4 mutations. 9 – 11 Emerging evidence indicates that the progression of AS shares pathological mechanisms with other CKDs, including inflammation, fibrosis, oxidative stress, and mitochondrial dysfunction. 12 Deficiencies or dysfunction in these collagen chains compromise GBM integrity and impair its filtration capacity, leading to proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis accompanied by inflammatory infiltration. 13 Regarding anemia in CKD, multiple mechanisms have been reported, the most prominent being: (1) reduced erythropoietin production, 14 (2) uremic toxin–induced impairment of erythropoiesis, 15 (3) increased eryptosis (premature red blood cell [RBC] destruction), 16 (4) inflammatory cytokine effects, 17 and (5) hepcidin-mediated disruption of iron metabolism. 18 However, none of the cited studies directly identify a mechanism for anemia in Alport syndrome. Although several studies have focused primarily on kidney disease progression, glomerular basement membrane alterations, and genetic mutations in collagen IV genes, 19 as well as complex pathological processes such as glomerular capillary distension 20 and mesangial cell invasion, 21 they have not explored the mechanisms underlying anemia. Previous knockout models of Col4a3 or Col4a5 recapitulated renal disease but were less translatable to the human condition, which arises from specific genetic variants rather than complete gene loss. To address this limitation, we previously developed a novel AS model carrying a patient-derived nonsense mutation (c.1411C > T, p.R471*) in exon 21 of Col4a5 , which mimics a known human XLAS mutation. 22 The phenotypic features of this model mirror those of human AS, including progressive proteinuria, glomerulosclerosis, and renal fibrosis; however, anemia has not yet been verified in these mice. In terms of pharmacological therapy, the most effective currently available treatments are renin–angiotensin system (RAS) inhibitors, including angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs). 23 ARBs reduce intraglomerular pressure and protect the glomerular endothelium and podocytes, leading to decreased urinary albumin excretion. 24 Several studies using mutant mouse models have demonstrated the efficacy of renin–angiotensin–aldosterone system (RAAS) inhibition with ACEIs or ARBs in treating Alport syndrome. 20 ˒ 25 However, no reports have demonstrated the renal ameliorative effects of RAAS inhibition in Col4a3– 5 mutation–based Alport syndrome mouse models. In the present study, we utilized the Col4a5 R471* mutant mouse to evaluate the therapeutic efficacy of irbesartan in AS, with particular focus on anemia. Methods Animals All mice were housed in a temperature- and humidity-controlled facility with ad libitum access to food and water and maintained under a 12-hour light–dark cycle (lights on 07:00–19:00). All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shonan Health Innovation Park, iPark Institute Co., Ltd., which is accredited by AAALAC International, and were conducted in accordance with institutional guidelines. All animal experiments were performed using male mice and approved by the Institutional Animal Care and Use Committee of the Shonan Research Center. The study was conducted and reported in accordance with the ARRIVE guidelines. Generation of Col4a5 R471* mutant mice The generation of Col4a5 R471* mutant mice was performed as described by Yamaura et al. 22 Briefly, a single-guide RNA and a single-stranded oligodeoxynucleotide were designed to introduce a mutation that produces a premature stop codon in exon 21 (c.1411C > T, p.R471*) of Col4a5 . Male Col4a5 R471* mutant and wild-type mice were obtained through natural mating. Phenotype analysis of Col4a5 R471* mutant mice At the start of the study, mice were 11 weeks of age and had body weights ranging from 23.0 to 28.6 g. Body weights were monitored throughout the experimental period to ensure comparable baseline conditions across groups. Plasma samples were collected from the tail vein at 11, 19, and 24 weeks of age. At 26 weeks of age, all mice were anesthetized with 3–5% isoflurane using an anesthetic vaporizer (ISOREX I-200, SHIN-EI INDUSTRIES, INC., Japan). Once a deep anesthetic state was confirmed based on the absence of the pain reflex, whole blood was collected from the abdominal vena cava, and the animals were euthanized by exsanguination. Plasma was isolated by centrifugation at 15,000 × g for 5 minutes. Blood urea nitrogen (BUN) was measured using the Clinical Analyzer 7180 (Hitachi High-Technologies, Tokyo, Japan). Urine samples were collected in metabolic cages at 12, 19, and 25 weeks of age to determine urine volume, albumin, and creatinine levels, and the urine albumin–creatinine ratio (UACR) was calculated. Urinary albumin was quantified using the Mouse Albumin ELISA Kit (Bethyl Laboratories Inc., USA). GFR analysis Male Col4a5 R471* mutant mice (14–15 weeks of age, n = 11; 25 weeks of age, n = 10) and wild-type C57BL/6J mice (14–15 weeks of age, n = 5) were used for glomerular filtration rate (GFR) analysis. GFR was determined by fluorescein isothiocyanate (FITC)–sinistrin clearance using transcutaneous measurement, as described previously. 26 ˒ 27 Briefly, FITC–sinistrin (50 mg/kg body weight, dissolved in sterile saline) was administered by retro-orbital injection. After injection, plasma samples were collected from the tail vein at 3, 7, 15, 35, 55, and 75 minutes. Plasma FITC–sinistrin concentrations were determined by comparing fluorescence intensities of standard curves and plasma samples using a microplate reader (EnVision). GFR was calculated from the plasma FITC–sinistrin concentration–time profiles using a two-phase decay model and expressed as mL/min/100 g body weight. Blood chemistry analysis Male Col4a5 R471* mutant mice and wild-type littermates were anesthetized with 3–5% isoflurane, and whole blood was collected from the abdominal vena cava at 22 weeks of age. RBC count, hemoglobin concentration, hematocrit value, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were measured using the ADVIA 2120i Hematology System (Siemens Healthcare Diagnostics, Japan). Repeated dosing study of Irbesartan Male Col4a5 R471* mutant mice generated by natural breeding were used in this study. Mice were enrolled at 5 weeks of age, and age-matched wild-type littermates served as controls. At 7 weeks of age, mice were acclimatized and allocated to experimental groups based on UACR and body weight. The body weights of the mice ranged from 20.5 to 23.7 g. Irbesartan was administered orally at a dose of 50 mg/kg once daily, and treatment continued for up to 17 weeks. Mice were monitored longitudinally with evaluations at baseline (pretreatment, 6 weeks of age) and at 5, 10, and 16 weeks after treatment initiation (corresponding to 12, 17, and 23 weeks of age, respectively). Body weight was measured weekly. Spot urine samples were collected at each evaluation point for measurement of urinary creatinine, albumin, and neutrophil gelatinase–associated lipocalin (NGAL). Urinary albumin and NGAL concentrations were determined using the Mouse Albumin ELISA Kit (Bethyl Laboratories Inc., USA) and the Mouse Lipocalin-2/NGAL Quantikine ELISA Kit (R&D Systems, USA), respectively, while creatinine was measured enzymatically using the Clinical Analyzer 7180 (Hitachi High-Technologies, Tokyo, Japan). UACR was then calculated. At 24 weeks of age, after 17 weeks of treatment, mice were anesthetized with 3–5% isoflurane, and whole blood was collected from the abdominal vena cava. RBC count, hemoglobin concentration, hematocrit value, MCV, and MCH were measured as indices of anemia using the ADVIA 120 Hematology System (Siemens Healthcare Diagnostics, Japan). At termination, kidneys were harvested for RNA extraction using the RNeasy Mini Kit (Qiagen, Tokyo, Japan). Complementary DNA (cDNA) was synthesized using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Tokyo, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using TaqMan Universal Master Mix II (Invitrogen, Tokyo, Japan) and the ABI 7900 system (Life Technologies, Tokyo, Japan), following the manufacturer’s instructions. The sets of quantitative real-time PCR probes used were as follows: tumor necrosis factor ( Tnf ; Mm00443258), interleukin-1 beta ( Il1b ; Mm00434228), interleukin-6 ( Il6 ; Mm00446190), C-C motif chemokine ligand 2 ( Ccl2 ; Mm00441242), F4/80 ( Adgre1 ; Mm00802529), NGAL ( Lcn2 ; Mm01324470), kidney injury molecule-1 (KIM-1; Havcr1 ; Mm00506686), collagen type I alpha 1 ( Col1a1 ; Mm00801666), alpha–smooth muscle actin ( Acta2 ; Mm00725412), and transforming growth factor beta 1 (TGF-β1; Tgfb1 ; Mm01178820). Glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ; Mm99999915) was used as the endogenous control gene. Relative gene expression levels were calculated using the 2 –ΔΔCt method. Statistical Analysis Longitudinal data are presented as the mean ± standard deviation (SD), whereas other data are shown as box-and-whisker plots. Statistical comparisons between wild-type and Col4a5 R471* mutant mice, as well as between vehicle- and irbesartan-treated mutant mice, were performed using Student’s t-test or the Wilcoxon rank-sum test, as appropriate. Comparisons among multiple treatment groups were analyzed by one-way analysis of variance, followed by post hoc testing. A p-value < 0.05 was considered statistically significant. Results Phenotype analysis of Col4a5 R471* mutant mice and kidney function Phenotypic analysis demonstrated marked renal impairment and pathological alterations in Col4a5 R471* mutant mice compared with age-matched wild-type controls. Body weight did not differ at early stages but tended to decrease with disease progression. Renal injury markers, including urine volume, UACR, and plasma BUN, were significantly increased with age (Fig. 1 A–D). To determine GFR, mice were injected with FITC–sinistrin, and blood samples were collected at multiple time points. Compared with wild-type mice, FITC–sinistrin remained at higher plasma concentrations in Col4a5 R471* mutant mice, and the calculated GFR was significantly reduced (Fig. 1 E and F). Phenotype analysis of Col4a5 R471* mutant mice and anemia At 22 weeks of age, blood samples were collected from wild-type and Col4a5 R471* mutant mice for hematological analysis. RBC count, hemoglobin concentration, hematocrit value, MCV, and MCH were all significantly reduced in Col4a5 R471* mutant mice compared with wild-type controls (Fig. 2 A–E), indicating anemia similar to that observed in patients with AS. Efficacy of irbesartan treatment on kidney function We subsequently evaluated the efficacy of irbesartan in the AS mouse model. Mice were treated with irbesartan for 17 weeks starting at 7 weeks of age. Both body weight and kidney weight were significantly lower in Col4a5 R471* mutant mice than in wild-type mice (Fig. 3 A and B), and irbesartan treatment for 17 weeks did not alter these parameters. The area under the curve (AUC) for UACR during the experimental period was elevated in Col4a5 R471* mutant mice. At 23 weeks of age, urine volume and urinary NGAL levels were significantly increased, whereas urine creatinine levels were significantly decreased in Col4a5 R471* mutant mice compared with wild-type mice. Irbesartan treatment significantly suppressed the AUC of UACR, urine volume, and urinary NGAL levels compared with the vehicle-treated group, but did not alter urine creatinine levels (Fig. 3 C–F). Efficacy of irbesartan treatment on anemia In terms of hematological parameters, consistent with the findings shown in Fig. 2 , RBC count, hemoglobin concentration, hematocrit value, MCV, and MCH were significantly lower in Col4a5 R471* mutant mice than in wild-type mice at 24 weeks of age. Irbesartan treatment significantly improved blood hemoglobin concentration and MCV and tended to restore RBC count, hematocrit, and MCH levels compared with wild-type mice. Therefore, irbesartan ameliorated anemia in Col4a5 R471* mutant mice (Fig. 4 A–E). Efficacy of irbesartan treatment on kidney injury Regarding renal injury parameters, the expression levels of genes such as Tnf , Il1b , Il6 , Ccl2 , Adgre1 (F4/80), Lcn2 (NGAL), Havcr1 (KIM-1), Col1a1 , Acta2 (α-SMA), and Tgfb1 in the kidneys of Col4a5 R471* mutant mice were significantly upregulated compared with those in wild-type mice (Fig. 5 A–G). Irbesartan treatment significantly suppressed or tended to suppress the expression of proinflammatory genes such as Tnf , Ccl2 , Adgre1 (F4/80), Il1b , and Il6 , as well as renal injury–related Lcn2 (NGAL) and Havcr1 (KIM-1) mRNA levels. However, fibrosis-related genes including Col1a1 , Acta2 , and Tgfb1 were not significantly affected by irbesartan treatment (Fig. 5 A–G). Discussion We performed a comprehensive pathological characterization of Col4a5 R471* mutant mice carrying a mutation identical to that observed in patients with Alport syndrome. 28 Consistent with previous reports, these mice exhibited age-dependent increases in UACR, urine volume, urinary kidney injury markers such as NGAL, and plasma BUN levels, indicating progressive renal dysfunction with age. However, GFR and anemia had not been previously evaluated. In this study, we measured GFR longitudinally in Col4a5 R471* mutant mice and, for the first time, demonstrated a progressive decline in GFR with age (Fig. 1 ). Taken together, these findings indicate that Col4a5 R471* mutant mice recapitulate multiple key features of Alport syndrome, including impaired GFR and renal anemia. Although RAS inhibitors are considered the first-line therapy for Alport syndrome, 23 their therapeutic efficacy had not been previously validated in Col4a5 R471* mutant mice. Based on these considerations, we evaluated the effects of the ARB irbesartan. Irbesartan treatment significantly ameliorated UACR, urinary NGAL, and renal inflammatory gene expression. Several reports have shown that ARBs exert multiple mechanisms for ameliorating kidney injury, supported by robust evidence from both clinical and preclinical studies. H. Kobori et al. reported that ARBs reduce proteinuria by decreasing intraglomerular pressure, which results from improved renal blood flow and reduced ischemia. 24 Kaori Hayashi et al. demonstrated that the ameliorative effects were mediated through epigenetic reprogramming of podocyte gene expression, particularly by restoring nephrin expression. 29 A. Mii et al. showed that protection of glomerular cells through angiotensin receptor modulation plays an important role in ameliorating CKD. 30 Consistent with clinical findings showing that RAAS blockade delays end-stage renal disease (ESRD) progression in patients with Alport syndrome, 31 our study demonstrated that irbesartan suppressed the increase in the UACR in Col4a5 R471* mutant mice. Furthermore, we observed a decrease in proinflammatory gene expression in the kidneys of these mice. To date, no studies have specifically reported on anemia in Alport syndrome. Previous studies have reported modest reductions in hemoglobin levels following treatment with RAS inhibitors, particularly ACE inhibitors, and this effect has been attributed to altered erythropoietin signaling or interference with angiotensin II–mediated erythropoiesis. 32 ˒ 33 However, these effects appear to be context-dependent and may differ between ACE inhibitors and ARBs. It is well established that both angiotensin II receptor subtypes—AT1R and AT2R—are involved in the regulation of hematopoiesis. The bone marrow (BM) stromal microenvironment expresses AT1R and AT2R, through which the major effector of the renin–angiotensin system, angiotensin II (Ang II), exerts regulatory effects on hematopoietic activity in coordination with the BM microenvironment. 34 ACE inhibitors reduce Ang II production and thereby inhibit signaling through both AT1R and AT2R, whereas ARBs selectively and more strongly inhibit AT1R signaling. 35 In clinical settings, accumulating evidence suggests that ARBs exert a smaller impact on hemoglobin levels than ACE inhibitors. 36 In the present study, irbesartan treatment was associated with improvements in anemia-related parameters in Col4a5 R471* mutant mice, suggesting that under conditions of progressive renal injury and inflammation, ARB-mediated renoprotection may outweigh potential suppressive effects on erythropoiesis. These findings indicate that the impact of ARB treatment on anemia should be interpreted within the context of underlying disease mechanisms rather than considered a uniform class effect. Further studies are warranted to establish a direct mechanistic link. Because our Col4a5 R471* mutant mice exhibit progressive GFR decline and renal anemia, they represent a valuable model for investigating disease-modifying therapies for CKD beyond Alport syndrome. A limitation of the present study is that we did not directly assess changes in GFR following irbesartan treatment in the Col4a5 R471* mutant model. Accurate measurement of GFR in mice requires repeated assessments within a short time frame, which imposes substantial procedural stress and can influence physiological parameters, particularly in fragile disease models. For this reason, and to minimize experimental burden, GFR measurement was not included as a primary endpoint in the current study and would require an independent experimental cohort. Instead, we focused on albuminuria and hematological parameters as functional readouts, which are clinically relevant and commonly used surrogate markers of disease progression in both experimental and clinical Alport syndrome. Importantly, irbesartan consistently improved albuminuria and anemia in this model, indicating beneficial effects on functional outcomes even in the absence of demonstrable structural remodeling. Future studies using appropriately powered cohorts will be necessary to determine whether the observed improvements in functional and hematological parameters are accompanied by measurable changes in GFR, and to further delineate the relationship between renal hemodynamics, filtration capacity, and extra-renal complications such as anemia in patient-relevant Col4a5 models. In summary, this study demonstrates that Col4a5 R471* mutant mice faithfully recapitulate the major clinical and molecular features of Alport syndrome, including progressive renal dysfunction, GFR decline, renal anemia, and tubulointerstitial injury. Irbesartan exerted pronounced renoprotective effects, underscoring the importance of renal hemodynamic improvement for disease modification. Declarations Acknowledgments The authors thank all laboratory members for their cooperation and technical assistance throughout this study. The authors also thank Dr. Masayuki Goto for his valuable advice and insightful discussions. Authorship contribution statement Takumi Sugawara: Writing – Original draft, Visualization, Methodology, Conduct research, Data Analysis and plan experiments, Mitsuharu Matsumoto- Methodology, Conduct research, Data Analysis and plan experiments, Masami Yamamoto, Masashi Aoyama, Nobuyuki Seki, Takako Iwachido, Kenta Danbayashi - Methodology, Conduct research, Data Analysis, Manami Kaneko-Supervision, Data Analysis, Methodology, Yasunori Nio-Writing, review & editing, Investigation, Methodology, Supervision, Investigation, Conceptualization. Data availability All data supporting the findings of this study are included within this article. The raw data generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could have influenced the work reported in this study. Ethical approval All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shonan Health Innovation Park, iPark Institute Co., Ltd., accredited by AAALAC International, and conducted in accordance with institutional guidelines. All animal experiments were conducted using male mice and approved by the Institutional Animal Care and Use Committee of Shonan Research Center (AU-00031347). References Gibson, J. et al. Prevalence estimates of predicted pathogenic COL4A3-COL4A5 variants in a population sequencing database and their implications for Alport syndrome. J. Am. Soc. Nephrol. 32 (9), 2273–2290. 10.1681/ASN.2020071065 (2021). Pajari, H., Kääriäinen, H., Muhonen, T. & Koskimies, O. Alport’s syndrome in 78 patients: Epidemiological and clinical study. 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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-8786563","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":600734482,"identity":"eb41d376-2e58-4500-b64e-bfd3dcd66baf","order_by":0,"name":"Takumi Sugawara","email":"","orcid":"","institution":"Axcelead Drug Discovery Partners, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Takumi","middleName":"","lastName":"Sugawara","suffix":""},{"id":600734483,"identity":"d2b5b21d-9dde-4dde-a0bd-f11bdb14b9c6","order_by":1,"name":"Mitsuharu Matsumoto","email":"","orcid":"","institution":"Axcelead Drug Discovery Partners, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Mitsuharu","middleName":"","lastName":"Matsumoto","suffix":""},{"id":600734484,"identity":"6836a006-42e6-46b1-a611-ddc31d139bb0","order_by":2,"name":"Masami Yamamoto","email":"","orcid":"","institution":"Axcelead Drug Discovery Partners, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Masami","middleName":"","lastName":"Yamamoto","suffix":""},{"id":600734485,"identity":"44283764-7bee-4e34-ba86-e69de294e1d8","order_by":3,"name":"Masashi Aoyama","email":"","orcid":"","institution":"Axcelead Drug Discovery Partners, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Masashi","middleName":"","lastName":"Aoyama","suffix":""},{"id":600734486,"identity":"bad347d2-2cfe-4134-8517-cb61f58363d1","order_by":4,"name":"Nobuyuki Seki","email":"","orcid":"","institution":"Axcelead Drug Discovery Partners, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Nobuyuki","middleName":"","lastName":"Seki","suffix":""},{"id":600734488,"identity":"a2f49639-ef8e-44af-9d54-1d6679bc9547","order_by":5,"name":"Takako Iwachido","email":"","orcid":"","institution":"Axcelead Drug Discovery Partners, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Takako","middleName":"","lastName":"Iwachido","suffix":""},{"id":600734489,"identity":"2e6c1301-4c81-45f0-ac66-ef61ba446487","order_by":6,"name":"Kenta Danbayashi","email":"","orcid":"","institution":"Axcelead Drug Discovery Partners, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Kenta","middleName":"","lastName":"Danbayashi","suffix":""},{"id":600734490,"identity":"d8e09850-e77b-4708-b067-eb1f0589c042","order_by":7,"name":"Manami Kaneko","email":"","orcid":"","institution":"Axcelead Drug Discovery Partners, Inc.","correspondingAuthor":false,"prefix":"","firstName":"Manami","middleName":"","lastName":"Kaneko","suffix":""},{"id":600734491,"identity":"89711ef5-ba70-4696-b0e7-112f9280e98b","order_by":8,"name":"Yasunori Nio","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYBADHgYGxgaGDyAmO4RLnBbGGSAmM5FawICZhxgtBsePP5MuqLgnwyDd3PjYpuawPD8z88EPbxjs5HQbcGg5k2MmPeNMMQ+DzMFm45xjaYYzm9mSJecwJBubHcCh5UAOmzRvWwIPg0Rim3QOm02CwWEeA2kehgOJ23BpOf/8GUKLxT+JBPvD/J9/49VyI8EMoYWxDWgLMw8bXlskb7wxtuY5k8DDJpHYbNjbl2Y44zCbmeUcA9x+4Tuf/vA2T0WCPb9E+sMHP74BQ6y9+fGNNxV2cri0KMDE2dAcjF05CMg34JYbBaNgFIyCUQABAO32UZAFxYzYAAAAAElFTkSuQmCC","orcid":"","institution":"Axcelead Drug Discovery Partners, Inc.","correspondingAuthor":true,"prefix":"","firstName":"Yasunori","middleName":"","lastName":"Nio","suffix":""}],"badges":[],"createdAt":"2026-02-04 12:38:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8786563/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8786563/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104402985,"identity":"506dd0ef-a3c5-4e0a-8ded-562dede0f4c7","added_by":"auto","created_at":"2026-03-11 12:17:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":623199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCol4a5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e R471* mutant mice for kidney function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A–D) Changes in body weight, plasma BUN, urine volume, and UACR over the study duration. Open circles indicate wild-type mice, and filled circles indicate \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice. (E) Plasma FITC–sinistrin concentration following retro-orbital (r.o.) injection of FITC–sinistrin. Open circles indicate wild-type mice at 14–15 weeks of age; gray circles indicate \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice at 14–15 weeks of age; filled circles indicate \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice at 25 weeks of age. (F) GFR of wild-type mice at 14–15 weeks of age and \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice at 14–15 and 25 weeks of age. Data for GFR are presented as box-and-whisker plots, while other data are shown as the mean ± standard deviation (SD). * p \u0026lt; 0.05, ** p \u0026lt; 0.01 vs. wild-type mice (Student’s t-test followed by Bonferroni correction). \u0026nbsp;\u003csup\u003e$$\u003c/sup\u003e p \u0026lt; 0.01, vs. wild-type mice (Dunnett’s test)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8786563/v1/dc8b834faf0e7178c53ef7ef.png"},{"id":104031367,"identity":"03b50b39-8e45-470a-b56b-8e33c18d13b8","added_by":"auto","created_at":"2026-03-06 00:26:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1051578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCol4a5\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e R471* mutant mice for anemia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole blood was collected from the abdominal vena cava at 22 weeks of age. (A) RBC count, (B) hemoglobin concentration, (C) hematocrit, (D) MCV, and (E) MCH were measured using an ADVIA hematology analyzer.\u003cbr\u003e\nData are presented as box-and-whisker plots.\u003c/p\u003e\n\u003cp\u003e* p \u0026lt; 0.05, ** p \u0026lt; 0.01 vs. wild-type mice (Student’s t-test)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8786563/v1/e1d4962a27551a3ddd287080.png"},{"id":104031365,"identity":"eb6c7530-f042-436b-ae9b-1a2b1f202504","added_by":"auto","created_at":"2026-03-06 00:26:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":793267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfficacy of irbesartan treatment on kidney function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Change in body weight over the study duration. (B) Total kidney weight at 24 weeks of age. (C) AUC of UACR over the study duration. (D–F) Urine volume, urinary NGAL, and urinary creatinine levels at 23 weeks of age. Data for body weight are presented as the mean ± standard deviation (SD), while other data are shown as box-and-whisker plots. * p \u0026lt; 0.05, ** p \u0026lt; 0.01 vs. wild-type mice (Student’s t-test followed by Bonferroni correction). \u003csup\u003e††\u003c/sup\u003e p \u0026lt; 0.01 vs. wild-type mice (Student’s t-test) \u003csup\u003e## \u003c/sup\u003ep \u0026lt; 0.01 vs. wild-type mice (Wilcoxon test).\u003csup\u003e $\u003c/sup\u003e p \u0026lt; 0.05,\u003csup\u003e $$\u003c/sup\u003e p \u0026lt; 0.01 vs. vehicle-treated group (Student’s t-test)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8786563/v1/733a049f52ffc9ac6758ed7d.png"},{"id":104031368,"identity":"2ea90d9e-9036-44dc-9f4b-94c7f1de8e47","added_by":"auto","created_at":"2026-03-06 00:26:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4029858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfficacy of irbesartan treatment on anemia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole blood was collected from the abdominal vena cava at 24 weeks of age. (A) RBC count, (B) hemoglobin concentration, (C) hematocrit, (D) MCV, and (E) MCH were measured using an ADVIA hematology analyzer. Data are presented as box-and-whisker plots.\u003c/p\u003e\n\u003cp\u003e** p \u0026lt; 0.01 vs. wild-type mice (Student’s t-test)\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e$\u003c/sup\u003e p \u0026lt; 0.05 vs. vehicle-treated group (Student’s t-test)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8786563/v1/2af4046e9f412149e60138f6.png"},{"id":104031363,"identity":"c2c2ce00-15dc-47d8-934a-49596b4bf3f4","added_by":"auto","created_at":"2026-03-06 00:26:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":827200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEfficacy of irbesartan treatment on kidney injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFold changes in mRNA expression levels of inflammation-, kidney injury–, and fibrosis-related genes in the kidney were normalized to those of the wild-type control group. Gapdh was used as an endogenous control. Data are presented as box-and-whisker plots. * p \u0026lt; 0.05, ** p \u0026lt; 0.01 vs. vehicle-treated group (Student’s t-test). \u003csup\u003e$ \u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e$$ \u003c/sup\u003ep \u0026lt; 0.01 vs. vehicle-treated group (Wilcoxon test)\u003c/p\u003e","description":"","filename":"floatimage51.png","url":"https://assets-eu.researchsquare.com/files/rs-8786563/v1/6c806beb91c34301a3109358.png"},{"id":106311296,"identity":"7a8dc670-2aef-4382-a0b8-d36b50d9fa59","added_by":"auto","created_at":"2026-04-07 10:28:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8223595,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8786563/v1/168aa664-7067-4752-8d7a-0ed02d7bab22.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Irbesartan ameliorates kidney injury and anemia in a Col4a5-mutant mouse model of Alport syndrome with CKD pathology","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlport syndrome (AS) is an inherited form of chronic kidney disease (CKD) characterized by progressive glomerular damage that ultimately leads to kidney failure and anemia. Its prevalence is estimated to range from 1 in 2,000 to 1 in 53,000 individuals, depending on diagnostic criteria and the extent of genetic screening.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Mutations in the type IV collagen genes\u0026mdash;\u003cem\u003eCOL4A3\u003c/em\u003e, \u003cem\u003eCOL4A4\u003c/em\u003e, and \u003cem\u003eCOL4A5\u003c/em\u003e\u0026mdash;cause structural abnormalities of the glomerular basement membrane (GBM), which contribute to the pathogenesis of Alport syndrome.\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e These genes encode the α3, α4, and α5 chains of type IV collagen, which are integral components of the mature GBM. A large-scale whole-exome sequencing study revealed that mutations in \u003cem\u003eCOL4A3\u003c/em\u003e, \u003cem\u003eCOL4A4\u003c/em\u003e, and \u003cem\u003eCOL4A5\u003c/em\u003e collectively account for nearly 30% of all monogenic CKD cases, establishing AS as the second most common hereditary nephropathy after autosomal dominant polycystic kidney disease.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis finding underscores the broader clinical relevance of AS, demonstrating that it is not only a rare disease but also a significant contributor to the overall burden of hereditary renal disorders. Approximately 80% of AS cases are X-linked (XLAS) and result from \u003cem\u003eCOL4A5\u003c/em\u003e mutations, whereas the remaining cases exhibit autosomal recessive or autosomal dominant inheritance due to \u003cem\u003eCOL4A3\u003c/em\u003e or \u003cem\u003eCOL4A4\u003c/em\u003e mutations.\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eEmerging evidence indicates that the progression of AS shares pathological mechanisms with other CKDs, including inflammation, fibrosis, oxidative stress, and mitochondrial dysfunction.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Deficiencies or dysfunction in these collagen chains compromise GBM integrity and impair its filtration capacity, leading to proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis accompanied by inflammatory infiltration.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Regarding anemia in CKD, multiple mechanisms have been reported, the most prominent being: (1) reduced erythropoietin production,\u003csup\u003e14\u003c/sup\u003e (2) uremic toxin\u0026ndash;induced impairment of erythropoiesis,\u003csup\u003e15\u003c/sup\u003e (3) increased eryptosis (premature red blood cell [RBC] destruction),\u003csup\u003e16\u003c/sup\u003e (4) inflammatory cytokine effects,\u003csup\u003e17\u003c/sup\u003e and (5) hepcidin-mediated disruption of iron metabolism.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e However, none of the cited studies directly identify a mechanism for anemia in Alport syndrome. Although several studies have focused primarily on kidney disease progression, glomerular basement membrane alterations, and genetic mutations in collagen IV genes,\u003csup\u003e19\u003c/sup\u003e as well as complex pathological processes such as glomerular capillary distension\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and mesangial cell invasion,\u003csup\u003e21\u003c/sup\u003e they have not explored the mechanisms underlying anemia.\u003c/p\u003e \u003cp\u003ePrevious knockout models of \u003cem\u003eCol4a3\u003c/em\u003e or \u003cem\u003eCol4a5\u003c/em\u003e recapitulated renal disease but were less translatable to the human condition, which arises from specific genetic variants rather than complete gene loss. To address this limitation, we previously developed a novel AS model carrying a patient-derived nonsense mutation (c.1411C\u0026thinsp;\u0026gt;\u0026thinsp;T, p.R471*) in exon 21 of \u003cem\u003eCol4a5\u003c/em\u003e, which mimics a known human XLAS mutation.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e The phenotypic features of this model mirror those of human AS, including progressive proteinuria, glomerulosclerosis, and renal fibrosis; however, anemia has not yet been verified in these mice.\u003c/p\u003e \u003cp\u003eIn terms of pharmacological therapy, the most effective currently available treatments are renin\u0026ndash;angiotensin system (RAS) inhibitors, including angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs).\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e ARBs reduce intraglomerular pressure and protect the glomerular endothelium and podocytes, leading to decreased urinary albumin excretion.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Several studies using mutant mouse models have demonstrated the efficacy of renin\u0026ndash;angiotensin\u0026ndash;aldosterone system (RAAS) inhibition with ACEIs or ARBs in treating Alport syndrome.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e˒\u003csup\u003e25\u003c/sup\u003e However, no reports have demonstrated the renal ameliorative effects of RAAS inhibition in \u003cem\u003eCol4a3\u0026ndash;\u003c/em\u003e5 mutation\u0026ndash;based Alport syndrome mouse models. In the present study, we utilized the \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mouse to evaluate the therapeutic efficacy of irbesartan in AS, with particular focus on anemia.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAll mice were housed in a temperature- and humidity-controlled facility with ad libitum access to food and water and maintained under a 12-hour light\u0026ndash;dark cycle (lights on 07:00\u0026ndash;19:00). All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shonan Health Innovation Park, iPark Institute Co., Ltd., which is accredited by AAALAC International, and were conducted in accordance with institutional guidelines. All animal experiments were performed using male mice and approved by the Institutional Animal Care and Use Committee of the Shonan Research Center. The study was conducted and reported in accordance with the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGeneration of Col4a5 R471* mutant mice\u003c/h3\u003e\n\u003cp\u003eThe generation of \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice was performed as described by Yamaura et al.\u003csup\u003e22\u003c/sup\u003e Briefly, a single-guide RNA and a single-stranded oligodeoxynucleotide were designed to introduce a mutation that produces a premature stop codon in exon 21 (c.1411C\u0026thinsp;\u0026gt;\u0026thinsp;T, p.R471*) of \u003cem\u003eCol4a5\u003c/em\u003e. Male \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant and wild-type mice were obtained through natural mating.\u003c/p\u003e\n\u003ch3\u003ePhenotype analysis of Col4a5 R471* mutant mice\u003c/h3\u003e\n\u003cp\u003eAt the start of the study, mice were 11 weeks of age and had body weights ranging from 23.0 to 28.6 g. Body weights were monitored throughout the experimental period to ensure comparable baseline conditions across groups. Plasma samples were collected from the tail vein at 11, 19, and 24 weeks of age. At 26 weeks of age, all mice were anesthetized with 3\u0026ndash;5% isoflurane using an anesthetic vaporizer (ISOREX I-200, SHIN-EI INDUSTRIES, INC., Japan). Once a deep anesthetic state was confirmed based on the absence of the pain reflex, whole blood was collected from the abdominal vena cava, and the animals were euthanized by exsanguination. Plasma was isolated by centrifugation at 15,000 \u0026times; g for 5 minutes. Blood urea nitrogen (BUN) was measured using the Clinical Analyzer 7180 (Hitachi High-Technologies, Tokyo, Japan). Urine samples were collected in metabolic cages at 12, 19, and 25 weeks of age to determine urine volume, albumin, and creatinine levels, and the urine albumin\u0026ndash;creatinine ratio (UACR) was calculated. Urinary albumin was quantified using the Mouse Albumin ELISA Kit (Bethyl Laboratories Inc., USA).\u003c/p\u003e\n\u003ch3\u003eGFR analysis\u003c/h3\u003e\n\u003cp\u003eMale \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice (14\u0026ndash;15 weeks of age, n\u0026thinsp;=\u0026thinsp;11; 25 weeks of age, n\u0026thinsp;=\u0026thinsp;10) and wild-type C57BL/6J mice (14\u0026ndash;15 weeks of age, n\u0026thinsp;=\u0026thinsp;5) were used for glomerular filtration rate (GFR) analysis. GFR was determined by fluorescein isothiocyanate (FITC)\u0026ndash;sinistrin clearance using transcutaneous measurement, as described previously.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e˒\u003csup\u003e27\u003c/sup\u003e Briefly, FITC\u0026ndash;sinistrin (50 mg/kg body weight, dissolved in sterile saline) was administered by retro-orbital injection. After injection, plasma samples were collected from the tail vein at 3, 7, 15, 35, 55, and 75 minutes. Plasma FITC\u0026ndash;sinistrin concentrations were determined by comparing fluorescence intensities of standard curves and plasma samples using a microplate reader (EnVision). GFR was calculated from the plasma FITC\u0026ndash;sinistrin concentration\u0026ndash;time profiles using a two-phase decay model and expressed as mL/min/100 g body weight.\u003c/p\u003e\n\u003ch3\u003eBlood chemistry analysis\u003c/h3\u003e\n\u003cp\u003eMale \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice and wild-type littermates were anesthetized with 3\u0026ndash;5% isoflurane, and whole blood was collected from the abdominal vena cava at 22 weeks of age. RBC count, hemoglobin concentration, hematocrit value, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were measured using the ADVIA 2120i Hematology System (Siemens Healthcare Diagnostics, Japan).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRepeated dosing study of Irbesartan\u003c/h2\u003e \u003cp\u003eMale \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice generated by natural breeding were used in this study. Mice were enrolled at 5 weeks of age, and age-matched wild-type littermates served as controls. At 7 weeks of age, mice were acclimatized and allocated to experimental groups based on UACR and body weight. The body weights of the mice ranged from 20.5 to 23.7 g. Irbesartan was administered orally at a dose of 50 mg/kg once daily, and treatment continued for up to 17 weeks. Mice were monitored longitudinally with evaluations at baseline (pretreatment, 6 weeks of age) and at 5, 10, and 16 weeks after treatment initiation (corresponding to 12, 17, and 23 weeks of age, respectively). Body weight was measured weekly. Spot urine samples were collected at each evaluation point for measurement of urinary creatinine, albumin, and neutrophil gelatinase\u0026ndash;associated lipocalin (NGAL). Urinary albumin and NGAL concentrations were determined using the Mouse Albumin ELISA Kit (Bethyl Laboratories Inc., USA) and the Mouse Lipocalin-2/NGAL Quantikine ELISA Kit (R\u0026amp;D Systems, USA), respectively, while creatinine was measured enzymatically using the Clinical Analyzer 7180 (Hitachi High-Technologies, Tokyo, Japan). UACR was then calculated. At 24 weeks of age, after 17 weeks of treatment, mice were anesthetized with 3\u0026ndash;5% isoflurane, and whole blood was collected from the abdominal vena cava. RBC count, hemoglobin concentration, hematocrit value, MCV, and MCH were measured as indices of anemia using the ADVIA 120 Hematology System (Siemens Healthcare Diagnostics, Japan). At termination, kidneys were harvested for RNA extraction using the RNeasy Mini Kit (Qiagen, Tokyo, Japan). Complementary DNA (cDNA) was synthesized using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Tokyo, Japan) according to the manufacturer\u0026rsquo;s instructions. Quantitative real-time PCR was performed using TaqMan Universal Master Mix II (Invitrogen, Tokyo, Japan) and the ABI 7900 system (Life Technologies, Tokyo, Japan), following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eThe sets of quantitative real-time PCR probes used were as follows: tumor necrosis factor (\u003cem\u003eTnf\u003c/em\u003e; Mm00443258), interleukin-1 beta (\u003cem\u003eIl1b\u003c/em\u003e; Mm00434228), interleukin-6 (\u003cem\u003eIl6\u003c/em\u003e; Mm00446190), C-C motif chemokine ligand 2 (\u003cem\u003eCcl2\u003c/em\u003e; Mm00441242), F4/80 (\u003cem\u003eAdgre1\u003c/em\u003e; Mm00802529), NGAL (\u003cem\u003eLcn2\u003c/em\u003e; Mm01324470), kidney injury molecule-1 (KIM-1; \u003cem\u003eHavcr1\u003c/em\u003e; Mm00506686), collagen type I alpha 1 (\u003cem\u003eCol1a1\u003c/em\u003e; Mm00801666), alpha\u0026ndash;smooth muscle actin (\u003cem\u003eActa2\u003c/em\u003e; Mm00725412), and transforming growth factor beta 1 (TGF-β1; \u003cem\u003eTgfb1\u003c/em\u003e; Mm01178820). Glyceraldehyde-3-phosphate dehydrogenase (\u003cem\u003eGapdh\u003c/em\u003e; Mm99999915) was used as the endogenous control gene. Relative gene expression levels were calculated using the 2\u003csup\u003e\u0026ndash;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eLongitudinal data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), whereas other data are shown as box-and-whisker plots. Statistical comparisons between wild-type and \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice, as well as between vehicle- and irbesartan-treated mutant mice, were performed using Student\u0026rsquo;s t-test or the Wilcoxon rank-sum test, as appropriate. Comparisons among multiple treatment groups were analyzed by one-way analysis of variance, followed by post hoc testing. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePhenotype analysis of\u003c/b\u003e \u003cb\u003eCol4a5\u003c/b\u003e \u003cb\u003eR471* mutant mice and kidney function\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePhenotypic analysis demonstrated marked renal impairment and pathological alterations in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice compared with age-matched wild-type controls. Body weight did not differ at early stages but tended to decrease with disease progression. Renal injury markers, including urine volume, UACR, and plasma BUN, were significantly increased with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine GFR, mice were injected with FITC\u0026ndash;sinistrin, and blood samples were collected at multiple time points. Compared with wild-type mice, FITC\u0026ndash;sinistrin remained at higher plasma concentrations in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice, and the calculated GFR was significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhenotype analysis of\u003c/b\u003e \u003cb\u003eCol4a5\u003c/b\u003e \u003cb\u003eR471* mutant mice and anemia\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAt 22 weeks of age, blood samples were collected from wild-type and \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice for hematological analysis. RBC count, hemoglobin concentration, hematocrit value, MCV, and MCH were all significantly reduced in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice compared with wild-type controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;E), indicating anemia similar to that observed in patients with AS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy of irbesartan treatment on kidney function\u003c/h2\u003e \u003cp\u003eWe subsequently evaluated the efficacy of irbesartan in the AS mouse model. Mice were treated with irbesartan for 17 weeks starting at 7 weeks of age. Both body weight and kidney weight were significantly lower in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice than in wild-type mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B), and irbesartan treatment for 17 weeks did not alter these parameters. The area under the curve (AUC) for UACR during the experimental period was elevated in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice. At 23 weeks of age, urine volume and urinary NGAL levels were significantly increased, whereas urine creatinine levels were significantly decreased in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice compared with wild-type mice. Irbesartan treatment significantly suppressed the AUC of UACR, urine volume, and urinary NGAL levels compared with the vehicle-treated group, but did not alter urine creatinine levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy of irbesartan treatment on anemia\u003c/h2\u003e \u003cp\u003eIn terms of hematological parameters, consistent with the findings shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, RBC count, hemoglobin concentration, hematocrit value, MCV, and MCH were significantly lower in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice than in wild-type mice at 24 weeks of age. Irbesartan treatment significantly improved blood hemoglobin concentration and MCV and tended to restore RBC count, hematocrit, and MCH levels compared with wild-type mice. Therefore, irbesartan ameliorated anemia in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEfficacy of irbesartan treatment on kidney injury\u003c/h2\u003e \u003cp\u003eRegarding renal injury parameters, the expression levels of genes such as \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eIl1b\u003c/em\u003e, \u003cem\u003eIl6\u003c/em\u003e, \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eAdgre1\u003c/em\u003e (F4/80), \u003cem\u003eLcn2\u003c/em\u003e (NGAL), \u003cem\u003eHavcr1\u003c/em\u003e (KIM-1), \u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003eActa2\u003c/em\u003e (α-SMA), and \u003cem\u003eTgfb1\u003c/em\u003e in the kidneys of \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice were significantly upregulated compared with those in wild-type mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;G). Irbesartan treatment significantly suppressed or tended to suppress the expression of proinflammatory genes such as \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eAdgre1\u003c/em\u003e (F4/80), \u003cem\u003eIl1b\u003c/em\u003e, and \u003cem\u003eIl6\u003c/em\u003e, as well as renal injury\u0026ndash;related \u003cem\u003eLcn2\u003c/em\u003e (NGAL) and \u003cem\u003eHavcr1\u003c/em\u003e (KIM-1) mRNA levels. However, fibrosis-related genes including \u003cem\u003eCol1a1\u003c/em\u003e, \u003cem\u003eActa2\u003c/em\u003e, and \u003cem\u003eTgfb1\u003c/em\u003e were not significantly affected by irbesartan treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe performed a comprehensive pathological characterization of \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice carrying a mutation identical to that observed in patients with Alport syndrome.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Consistent with previous reports, these mice exhibited age-dependent increases in UACR, urine volume, urinary kidney injury markers such as NGAL, and plasma BUN levels, indicating progressive renal dysfunction with age. However, GFR and anemia had not been previously evaluated. In this study, we measured GFR longitudinally in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice and, for the first time, demonstrated a progressive decline in GFR with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Taken together, these findings indicate that \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice recapitulate multiple key features of Alport syndrome, including impaired GFR and renal anemia. Although RAS inhibitors are considered the first-line therapy for Alport syndrome,\u003csup\u003e23\u003c/sup\u003e their therapeutic efficacy had not been previously validated in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice. Based on these considerations, we evaluated the effects of the ARB irbesartan. Irbesartan treatment significantly ameliorated UACR, urinary NGAL, and renal inflammatory gene expression.\u003c/p\u003e \u003cp\u003eSeveral reports have shown that ARBs exert multiple mechanisms for ameliorating kidney injury, supported by robust evidence from both clinical and preclinical studies. H. Kobori et al. reported that ARBs reduce proteinuria by decreasing intraglomerular pressure, which results from improved renal blood flow and reduced ischemia.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Kaori Hayashi et al. demonstrated that the ameliorative effects were mediated through epigenetic reprogramming of podocyte gene expression, particularly by restoring nephrin expression.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e A. Mii et al. showed that protection of glomerular cells through angiotensin receptor modulation plays an important role in ameliorating CKD.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Consistent with clinical findings showing that RAAS blockade delays end-stage renal disease (ESRD) progression in patients with Alport syndrome,\u003csup\u003e31\u003c/sup\u003e our study demonstrated that irbesartan suppressed the increase in the UACR in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice. Furthermore, we observed a decrease in proinflammatory gene expression in the kidneys of these mice. To date, no studies have specifically reported on anemia in Alport syndrome.\u003c/p\u003e \u003cp\u003ePrevious studies have reported modest reductions in hemoglobin levels following treatment with RAS inhibitors, particularly ACE inhibitors, and this effect has been attributed to altered erythropoietin signaling or interference with angiotensin II\u0026ndash;mediated erythropoiesis.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e˒\u003csup\u003e33\u003c/sup\u003e However, these effects appear to be context-dependent and may differ between ACE inhibitors and ARBs.\u003c/p\u003e \u003cp\u003eIt is well established that both angiotensin II receptor subtypes\u0026mdash;AT1R and AT2R\u0026mdash;are involved in the regulation of hematopoiesis. The bone marrow (BM) stromal microenvironment expresses AT1R and AT2R, through which the major effector of the renin\u0026ndash;angiotensin system, angiotensin II (Ang II), exerts regulatory effects on hematopoietic activity in coordination with the BM microenvironment.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eACE inhibitors reduce Ang II production and thereby inhibit signaling through both AT1R and AT2R, whereas ARBs selectively and more strongly inhibit AT1R signaling.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e In clinical settings, accumulating evidence suggests that ARBs exert a smaller impact on hemoglobin levels than ACE inhibitors.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn the present study, irbesartan treatment was associated with improvements in anemia-related parameters in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice, suggesting that under conditions of progressive renal injury and inflammation, ARB-mediated renoprotection may outweigh potential suppressive effects on erythropoiesis. These findings indicate that the impact of ARB treatment on anemia should be interpreted within the context of underlying disease mechanisms rather than considered a uniform class effect. Further studies are warranted to establish a direct mechanistic link.\u003c/p\u003e \u003cp\u003eBecause our \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice exhibit progressive GFR decline and renal anemia, they represent a valuable model for investigating disease-modifying therapies for CKD beyond Alport syndrome.\u003c/p\u003e \u003cp\u003eA limitation of the present study is that we did not directly assess changes in GFR following irbesartan treatment in the \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant model. Accurate measurement of GFR in mice requires repeated assessments within a short time frame, which imposes substantial procedural stress and can influence physiological parameters, particularly in fragile disease models. For this reason, and to minimize experimental burden, GFR measurement was not included as a primary endpoint in the current study and would require an independent experimental cohort. Instead, we focused on albuminuria and hematological parameters as functional readouts, which are clinically relevant and commonly used surrogate markers of disease progression in both experimental and clinical Alport syndrome. Importantly, irbesartan consistently improved albuminuria and anemia in this model, indicating beneficial effects on functional outcomes even in the absence of demonstrable structural remodeling. Future studies using appropriately powered cohorts will be necessary to determine whether the observed improvements in functional and hematological parameters are accompanied by measurable changes in GFR, and to further delineate the relationship between renal hemodynamics, filtration capacity, and extra-renal complications such as anemia in patient-relevant \u003cem\u003eCol4a5\u003c/em\u003e models. In summary, this study demonstrates that \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice faithfully recapitulate the major clinical and molecular features of Alport syndrome, including progressive renal dysfunction, GFR decline, renal anemia, and tubulointerstitial injury. Irbesartan exerted pronounced renoprotective effects, underscoring the importance of renal hemodynamic improvement for disease modification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank all laboratory members for their cooperation and technical assistance throughout this study. The authors also thank Dr. Masayuki Goto for his valuable advice and insightful discussions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTakumi Sugawara:\u0026nbsp;Writing \u0026ndash; Original draft,\u0026nbsp;Visualization, Methodology, Conduct research, Data Analysis and plan experiments, Mitsuharu Matsumoto- Methodology, Conduct research, Data Analysis and plan experiments, Masami Yamamoto,\u0026nbsp;Masashi Aoyama, Nobuyuki Seki, Takako Iwachido, Kenta Danbayashi\u0026nbsp;-\u0026nbsp;Methodology,\u0026nbsp;Conduct research,\u0026nbsp;Data Analysis,\u0026nbsp;Manami Kaneko-Supervision,\u0026nbsp;Data\u0026nbsp;Analysis,\u0026nbsp;Methodology, Yasunori Nio-Writing,\u0026nbsp;review \u0026amp; editing, Investigation, Methodology, Supervision, Investigation, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are included within this article. The raw data generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships that could have influenced the work reported in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shonan Health Innovation Park, iPark Institute Co., Ltd., accredited by AAALAC International, and conducted in accordance with institutional guidelines. All animal experiments were conducted using male mice and approved by the Institutional Animal Care and Use Committee of Shonan Research Center (AU-00031347).\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGibson, J. et al. Prevalence estimates of predicted pathogenic COL4A3-COL4A5 variants in a population sequencing database and their implications for Alport syndrome. \u003cem\u003eJ. Am. Soc. Nephrol.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e (9), 2273\u0026ndash;2290. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1681/ASN.2020071065\u003c/span\u003e\u003cspan address=\"10.1681/ASN.2020071065\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePajari, H., K\u0026auml;\u0026auml;ri\u0026auml;inen, H., Muhonen, T. \u0026amp; Koskimies, O. 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Notes\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e, 443. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1756-0500-6-443\u003c/span\u003e\u003cspan address=\"10.1186/1756-0500-6-443\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alport syndrome, angiotensin receptor blocker, Irbesartan, anemia, collagen 4a5","lastPublishedDoi":"10.21203/rs.3.rs-8786563/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8786563/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlport syndrome, caused by mutations in type IV collagen genes, leads to progressive proteinuria, anemia, and glomerulosclerosis, ultimately resulting in renal failure. It also shares pathological features with chronic kidney disease (C KD). Previous knockout models of \u003cem\u003eCol4a3\u003c/em\u003e or \u003cem\u003eCol4a5\u003c/em\u003e recapitulated renal disease but were less translatable to the human condition, which arises from specific genetic variants rather than complete gene loss. To address this limitation, we established a \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mouse carrying a clinically relevant mutation and evaluated its responsiveness to the angiotensin receptor blocker (ARB) irbesartan as a benchmark for translational utility in ameliorating kidney injury and anemia. \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice exhibited significant increases in the urine albumin\u0026ndash;creatinine ratio (UACR) and blood urea nitrogen, along with age-dependent decreases in glomerular filtration rate. In addition, changes in anemia-related factors\u0026mdash;such as red blood cell count, hemoglobin level, and hematocrit value\u0026mdash;were observed. Irbesartan treatment significantly reduced UACR and urinary neutrophil gelatinase\u0026ndash;associated lipocalin levels and improved anemia-related factors and proinflammatory transcript expression. These results provide the first preclinical evidence that an ARB can improve kidney injury and anemia in \u003cem\u003eCol4a5\u003c/em\u003e R471* mutant mice, highlighting the model\u0026rsquo;s translational relevance for CKD drug evaluation.\u003c/p\u003e","manuscriptTitle":"Irbesartan ameliorates kidney injury and anemia in a Col4a5-mutant mouse model of Alport syndrome with CKD pathology","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-06 00:26:31","doi":"10.21203/rs.3.rs-8786563/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a357f744-ecb9-4c91-bdc1-29124f586d42","owner":[],"postedDate":"March 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63929138,"name":"Health sciences/Diseases"},{"id":63929139,"name":"Biological sciences/Genetics"},{"id":63929140,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2026-04-07T10:27:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-06 00:26:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8786563","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8786563","identity":"rs-8786563","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}