Divergent renal localization patterns of heterozygote-derived two distinct AA amyloids in a cat

Divergent renal localization patterns of heterozygote-derived two distinct AA amyloids in a cat | 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 Divergent renal localization patterns of heterozygote-derived two distinct AA amyloids in a cat Natsumi Kobayashi, Masahiro Kaneda, Susumu Iwaide, Yoshiyuki Itoh, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3865213/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted You are reading this latest preprint version Abstract Amyloid A (AA) amyloidosis poses a fatal threat to both humans and animals. While the kidneys represent the principal organ affected in AA amyloidosis, there exists variability in the localization of amyloid deposition, with distinct symptoms delineated by the specific deposition sites. Nevertheless, the factors contributing to the diversity of deposition remain unclear. In this study, we identified an association between serum amyloid A (SAA) polymorphisms and patterns of amyloid deposition. Histopathological analysis of the kidneys from a 5-year-old spayed female Japanese cat, which succumbed to systemic AA amyloidosis, revealed renal amyloid deposition in cortical glomeruli and medullary interstitium. Genetic analysis disclosed that the afflicted cat possessed a heterozygous SAA with three amino acid substitutions (K47I, Q63R, S93N), resulting in the SAA KQS and SAA IRN variants. Mass spectrometry and immunohistochemistry demonstrated that SAA KQS was deposited in the glomerulus and renal papilla, while SAA IRN was restricted to the extramedullary zone. This study established the differing renal distributions of two AA amyloid variants originating from heterozygotes within a single individual. The evidence supports the notion that the primary structure of precursor proteins defines the distribution of amyloid deposition. Health sciences/Pathogenesis Health sciences/Nephrology/Kidney diseases Biological sciences/Molecular biology/Protein folding/Protein aggregation Biological sciences/Biochemistry/Peptides Figures Figure 1 Figure 2 Introduction Systemic amyloidosis is a group of intractable diseases characterized by amyloid deposition in multiple organs due to misfolding of serum proteins 1 . Among systemic amyloidoses, amyloid A (AA) amyloidosis caused by serum amyloid A (SAA) is one of the most common diseases in humans and animals 2 , 3 . The kidneys are the primary target organ for AA amyloidosis, but the distribution of deposits varies from patient to patient. For example, in cattle, some individuals exhibit a glomerular predominant distribution of amyloid deposition, while others have predominant deposition in the medullary tubulointerstitium 4 . However, the factors responsible for the difference in deposition distribution have not been elucidated in animals. In humans, although glomerular involvement is the most common presentation of AA amyloidosis, some conditions with predominant deposition in vessels or tubules have also been described 3 , and these heterogeneous distributions have been explained by conformational differences resulting from different lengths of the SAA fragments that comprise the amyloid 5 . On the other hand, the diversity of AA amyloid distribution in the cortex and medulla is largely unexplored in humans because most analyses of renal amyloidosis in humans are based on cortical biopsy. The distribution of amyloid deposition within the kidney is closely related to clinical manifestations, especially glomerular deposition leads to lethal nephrotic syndrome 4 , Therefore, it is essential to elucidate what factors direct the amyloid distribution. In this study, we analyzed renal amyloid deposits in a cat expressing two heterozygous SAAs and found that the primary structure of SAAs affects the intrarenal distribution of AA amyloid deposition. Results Case Information The patient, a 4-year-old spayed female cat, presented to the authors' (MT and TA) veterinary clinic with the chief complaint of vomiting, loss of energy, and anorexia. The patient had previously received the feline immunodeficiency virus/feline leukemia virus test at another veterinary hospital was negative (test kit unknown). The patient had numerous ulcers in the oral cavity and a mass on the lower part of the tongue, which was treated with antibiotics and anti-inflammatory drugs. The mass in the hypoglossal area was removed under general anesthesia and submitted to a commercial pathology lab, which was histopathologically diagnosed as lymphoplasmacytic stomatitis with granulation tissue. About a year later, at age 5, in addition to the symptoms of gingivostomatitis, the patient began to exhibit polyuria and polydipsia, chronic renal insufficiency, and non-regenerative anemia. Therefore, she was treated with hematopoietic agents, oral medications for renal protection, and subcutaneous fluid replacement. Later, however, she showed gastrointestinal symptoms, then dehydration, further deterioration of renal function, hypothermia, loss of energy, anorexia, and severe non-regenerative anemia. Ultrasonography revealed mixed hypo- and hyperechoic nodules in the enlarged liver, so a fine needle aspiration of the liver was performed. Fine needle aspiration samples were submitted to a commercial pathology lab and cytologically diagnosed as hepatic amyloidosis. Despite various symptomatic treatments, including two blood transfusions and hematopoietic agents for severe anemia, antibiotics, anti-inflammatory drugs, and hepatoprotective drugs, the patient died 14 days after the cytological diagnosis of amyloidosis. Postmortem necropsy was performed promptly, and the liver and kidneys were sampled for histological examination. The liver was multinodularly enlarged, and blood clots were observed on the surface (Supplementary Fig. 1a). The kidney was slightly faded and had an uneven surface (Supplementary Fig. 1b). Histopathology Histologically, severe amyloid deposition was observed in the kidneys and liver (Fig. 1 a). In the kidney, amyloid deposits were widely observed in the cortical glomeruli and medullary interstitium, with particularly severe deposits in the outer medulla (Figs. 1 b- 1 d). SAA genes in the case Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing revealed eight heterozygous single nucleotide substitutions (c.111G > C, c.140A > T, c.183T > T, c.183T > C, c.188A > G, c.231T > C, c.252A > G, c.272G > A, c.282T > C) in the SAA gene resulting in three amino acid substitutions (K29I, Q45R, S75N). Cloning of PCR products revealed that the amino acid substitution combinations are Ile29-Arg45-Asn75 (SAA IRN ) and Lys29-Gln45-Ser75 (SAA KQS ; Fig. 2 a). BLAST analysis using the UniProt database showed that SAA KQS and SAA IRN were identical to entries M3WHE0 and A0A337SKP2, respectively. Distribution of heterozygote-derived amyloids To determine the respective renal distribution of amyloid derived from SAA IRN and SAA KQS , immunohistochemistry was performed using anti-SAA Q45 antibody, which specifically recognizes only SAA with Gln45, and anti-Pan-SAA antibody, which recognizes both SAAs with Gln45 or Arg45. All the renal amyloid deposits were positive for the anti-Pan-SAA antibody, indicating AA amyloidosis (Fig. 1 e). Immunohistochemistry using anti-SAA Q45 antibody was positive for amyloid deposits in the glomeruli, but mostly negative for the medullary deposits except in the papillae (Fig. 1 f). There were no noticeable distribution differences between the two antibodies in hepatic amyloid deposits (Supplementary Fig. 2). Mass spectrometry demonstrated that SAA is the major component of amyloid deposits in glomeruli, outer medulla, and papilla (Supplementary Table 1). Analysis of SAA-derived peptides detected by mass spectrometry revealed that N-terminal Gln1-Arg72-derived peptides were frequently detected in all three regions, while C-terminal peptides were rare (Figs. 2 b- 2 d). In all three regions, peptides with Ile29 were detected, but no peptides with Lys29 were detected. Focusing on the 45th amino acid residue, only Glu45 was detected in the glomeruli (Fig. 2 b), only Arg45 in the outer medulla (Fig. 2 c), and both Glu45 and Arg45 were in the papilla (Fig. 2 d). The partial overlap of the mass spectrometric results of amyloid collected from the papilla with those of the outer medulla is expected because the two were mixed during microdissection. Therefore, the following discussion will primarily focus on the glomerular and outer medullary results. Discussion Based on immunohistochemistry and mass spectrometry, this cat was diagnosed with AA amyloidosis. Genetic analysis showed that this cat had two heterozygous SAAs (SAA KQS , SAA IRN ), and immunohistochemistry and mass spectrometry further revealed that these two SAAs showed different distributions in nephron: SAA KQS -derived amyloid in the glomeruli and papilla, while SAA IRN -derived amyloid in the medulla. Functionally, the glomerulus is a mass of capillaries and produces primitive urine. Renal tubules in the medulla reabsorb the necessary components from primitive urine. The concentrated urine passes through the collecting duct and is released through the papillae into the pelvis. Since the pH and protein concentration in urine change dizzyingly as it passes through the nephrons, it may be natural that different biochemical properties of the amyloid would change the deposition site. This study has demonstrated that a few differences in the primary structure of amyloid precursor protein affect the intra-nephron distribution of amyloid deposition. This is the first report to demonstrate that two types of amyloid derived from heterozygotes show different distributions in a single individual. Notably, although the detection of Arg45 and Gln45 in the central region of SAA varied by deposition site, peptides containing Ile29 in the N-terminal region were consistently detected from each area. The protofilament polypeptides that make up the feline AA fibrils have 11 β-strands in an extended hairpin structure, with the Ile29 located on strand β4 at the beginning of the central β-arch; the hydrophobic side chain of Ile29 faces the inside of the β-arch and forms hydrophobic bonds with side chains of Pro48, Trp52, and Ala54 6 . Therefore, substituting the hydrophobic residue isoleucine for the basic residue lysine is expected to significantly affect the conformation around this region. Mass spectrometry analysis suggests that SAAs IRN can form amyloid by themself, but the central region of SAA KQS may acquire amyloidogenicity by interacting with the N-terminus of SAA IRN . The role of Ile29 in feline AA amyloidosis and the interaction between heterozygous SAAs should be investigated in the future. Conclusions This study provides evidence that differences in the primary structure of the precursor proteins affect the distribution of amyloid deposition by showing that heterozygote-derived two types of AA amyloid exhibit different renal distributions within a single individual. Different renal distribution of amyloid deposits produces different clinical manifestations; glomerular amyloid deposition causes proteinuria, while deposition in the tubular interstitium causes impaired urine concentration. 3 , 7 Future application of this study may enable the prediction of the AA amyloid-deposited site within the kidney and the prognosis by evaluating the SAA sequence of each patient. Materials and Methods Histopathological Analysis Portions of the liver and kidney were formalin-fixed and paraffin-embedded, cut into 3-µm sections, and stained with hematoxylin and eosin, and Congo red. Amyloid deposits were confirmed as Congo red-positive material showing yellow to green birefringence under polarized light. Sequencing of the SAA genes RT-PCR and sequencing were performed to determine the sequence of SAA . Primers designed using Primer Blast in NCBI based on the mRNA sequence of predicted Felis catus serum amyloid A (NCBI accession: XM_045038041.1) are as follows: forward, 5’-AGCTCTTCTCCACTGGGACT-3'; reverse, 5’-CTCTCCTGAGTGCAGAGCAA-3'. The total RNA was extracted from formalin-fixed paraffin-embedded liver tissue sections using the innuPREP FFPE total RNA Kit (Analytik Jena, Überlingen, Germany). One-step RT-PCR was performed using the PrimeScript One-Step RT-PCR Kit (Takara Bio, Shiga, Japan). RT-PCR was performed as follows: reverse transcription at 50°C for 30 min, 94°C for 30 s, 60°C for 30 s, 72°C for 1 min cycles 35 times with a final extension at 72°C for 3 min. Product size was analyzed using agarose gel electrophoresis, and target DNA fragments (446 bp) were purified from the gel using NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL, Düren, Germany). Nucleotide sequences were determined using the contract service of Eurofins Genomics (Tokyo, Japan). Each amino acid sequence was translated from the sequenced codon using EMBOSS Transeq 8 . Cloning and sequencing of heterozygous SAA The amplified PCR products were cloned using pGEM®-T Easy Vector Systems (Promega, Madison, WI, USA) and sent for a sequencing service (Greiner Bio-One, Frickenhausen, Germany). Amino acid sequences were translated from the sequenced codon using EMBOSS Transeq 8 . The determined SAA sequences were compared to the SAA sequences registered in UniProt by BLAST 9 . Immunohistochemistry A new antibody was generated for DKYFHARGNYDAAQRGPGG (corresponding to residues 32–50 of mature cat SAA), a sequence of SAA amino acid sequence that is well conserved in animals 10 , using the contract service of Cosmo Bio Co. (Tokyo, Japan). Target sequence synthetic peptide [C + DKYFHARGNYDAAQRGPGG] was immunized to rabbits, and their serum was obtained. In addition to the newly produced SAA antibody, a commercially available SAA antibody (PAA885Hu01, Cloud-Clone Corp., USA) was used as the primary antibody. The newly produced SAA antibody and the commercial SAA antibody are referred to as anti-SAAQ45 antibody and anti-Pan-SAA antibody, respectively, based on the verification of cross-reactivity described in Supplementary Methods and Supplementary Fig. 3. Horseradish peroxidase-conjugated polymer anti-rabbit IgG antibody (Dako, Santa Clara, CA, USA) was used as the secondary antibody, and a positive reaction was obtained with 3,3'-diaminobenzidine tetrahydrochloride. Normal rabbit serum was used instead of the primary antibody for the negative control. Tissue Microdissection and Mass Spectrometry Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of amyloid deposits collected from Congo red-stained sections was performed to determine the protein profile in the amyloid deposits. As in the previous study 11 , 12 , Congo red-positive amyloid deposits in glomeruli, outer medulla, and papillae were collected from five regions of approximately 500,000 µm 2 each under a stereomicroscope. Three samples of each collection were digested with trypsin, and two samples were digested with chymotrypsin before LC-MS/MS analysis, as described previously 11 , 12 . First, to clarify the protein composition in amyloid deposits, the MS/MS data of three tryptic samples were collated to theoretical fragment patterns of tryptic peptide sequences from the UniProt database using Mascot Server (Matrix Science Inc). The in silico proteolytic enzymes "Trypsin/P" in the Mascot Server's drop-down list were selected. Statistically significant proteins/peptides were extracted by Mascot's probability-based scoring algorithm. Next, MS/MS data were collated to two SAA sequences (SAA KQS and SAA IRN ) identified in this study to determine which of the heterozygous SAAs the amyloid deposited in each region derived from. The in silico enzymes "semiTrypsin" and "none" in the Mascot Server's drop-down list were selected to identify nontryptic and nonchymotryptic peptides, respectively. Statistically significant peptides were extracted using Mascot's probability-based scoring algorithm. The distribution of detected peptides was mapped in each SAA sequence. Declarations Acknowledgments This research was supported by JSPS KAKENHI (Grant No. 23H02380) and the Program on Open Innovation Platform with Enterprises, Research Institute, and Academia (OPERA) from the JST. Author contributions NK and TM conceptualized the paper. NK, MK, SI, YI, and MH performed the experiments and validated the study. NK, MK, and TM performed the formal analysis. NK, SI, YK, and NSM investigated. MT and TA recorded clinical matters and provided resources. NK and TM wrote the original draft and visualized the study. All authors reviewed and TM edited. MK, YI, and TM supervised. TM performed project administration and acquired funding. Data Availability All data are available in the main text or the Supplementary Materials. Please contact the corresponding author ( [email protected] ) for general comments or material requests. The data supporting the findings of this study can be provided upon reasonable request. Competing Interests Statement All the authors declared no competing interests. References Buxbaum, J. N. et al. Amyloid nomenclature 2022: update, novel proteins, and recommendations by the International Society of Amyloidosis (ISA) Nomenclature Committee. Amyloid 29, 213–219 (2022). Pinney, J. H. & Lachmann, H. J. Systemic AA Amyloidosis. in Protein Aggregation and Fibrillogenesis in Cerebral and Systemic Amyloid Disease (ed. Harris, J. R.) vol. 65 541–564 (Springer Netherlands, 2012). Karam, S., Haidous, M., Royal, V. & Leung, N. Renal AA amyloidosis: presentation, diagnosis, and current therapeutic options: a review. Kidney International 103, 473–484 (2023). Murakami, T. et al. Atypical AA amyloid deposits in bovine AA amyloidosis. Amyloid 19, 15–20 (2012). Banerjee, S. et al. Amyloid fibril structure from the vascular variant of systemic AA amyloidosis. Nat Commun 13, 7261 (2022). Schulte, T. et al. Cryo-EM structure of ex vivo fibrils associated with extreme AA amyloidosis prevalence in a cat shelter. Nat Commun 13, 7041 (2022). Khalighi, M. A., Dean Wallace, W. & Palma-Diaz, M. F. Amyloid nephropathy. Clinical Kidney Journal 7, 97–106 (2014). Madeira, F. et al. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res 50, W276–W279 (2022). The UniProt Consortium. UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Research 51, D523–D531 (2023). Lu, J., Yu, Y., Zhu, I., Cheng, Y. & Sun, P. D. Structural mechanism of serum amyloid A-mediated inflammatory amyloidosis. Proceedings of the National Academy of Sciences 111, 5189–5194 (2014). Murakami, T. et al. Identification of novel amyloidosis in dogs: α-S1-casein acquires amyloidogenicity in mammary tumor by overexpression and N-terminal truncation. Vet Pathol 60, 203–213 (2023). Iwaide, S. et al. Fibrinogen Aα-chain amyloidosis outbreaks in Japanese squirrels (Sciurus lis): a potential disease model. The Journal of Pathology 261, 96–104 (2023). Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials240115.docx Cite Share Download PDF Status: Published Journal Publication published 30 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-3865213","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":268181157,"identity":"75e81e2d-d085-4fee-ab91-d001e72c825b","order_by":0,"name":"Natsumi Kobayashi","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Natsumi","middleName":"","lastName":"Kobayashi","suffix":""},{"id":268181158,"identity":"32aa4a27-b378-4bf7-b139-5eeae86bbbab","order_by":1,"name":"Masahiro Kaneda","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Masahiro","middleName":"","lastName":"Kaneda","suffix":""},{"id":268181159,"identity":"e9d88100-3c00-4bcd-9392-485537f75f06","order_by":2,"name":"Susumu Iwaide","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Susumu","middleName":"","lastName":"Iwaide","suffix":""},{"id":268181160,"identity":"39f97ba9-b7d1-4bbc-b1b8-c2390ee2ac80","order_by":3,"name":"Yoshiyuki Itoh","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yoshiyuki","middleName":"","lastName":"Itoh","suffix":""},{"id":268181161,"identity":"e0ff4cb8-fc99-4a1f-acef-4ca82cedd229","order_by":4,"name":"Miki Hisada","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Miki","middleName":"","lastName":"Hisada","suffix":""},{"id":268181162,"identity":"a60071d4-e23a-44fc-8ca0-5db4b80d0520","order_by":5,"name":"Yuka Kato","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuka","middleName":"","lastName":"Kato","suffix":""},{"id":268181163,"identity":"06daa51f-68fc-47a1-83bf-4eabab861594","order_by":6,"name":"Niki Sedghi Masoud","email":"","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":false,"prefix":"","firstName":"Niki","middleName":"Sedghi","lastName":"Masoud","suffix":""},{"id":268181164,"identity":"97052b28-322f-4989-9988-55ceabba8e27","order_by":7,"name":"Machie Tsuneyasu","email":"","orcid":"","institution":"Watanabe Animal Hospital, Anicom Specialty Medical Institute Inc","correspondingAuthor":false,"prefix":"","firstName":"Machie","middleName":"","lastName":"Tsuneyasu","suffix":""},{"id":268181165,"identity":"9fd78324-bd98-49d1-8df1-f749a2418be5","order_by":8,"name":"Tomoko Akamine","email":"","orcid":"","institution":"Watanabe Animal Hospital, Anicom Specialty Medical Institute Inc","correspondingAuthor":false,"prefix":"","firstName":"Tomoko","middleName":"","lastName":"Akamine","suffix":""},{"id":268181166,"identity":"22cfea45-832c-4ffa-98b5-42432c66fd52","order_by":9,"name":"Tomoaki Murakami","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYFACNiCuQBE5QISWA2egWg8QreVgG4oWAkB39rHUzR/n2ckzyPcYfv7AAGQwnsWv0+xc2rEbB7clGzaw8RhLHGAAMhjOJeDXcoa9DajlQAIDG48BUAszUPkZAyK0zAFrMf5xgKGeGC1sQIc1gLWYAW05TJSWtBtnjiUbtrGllVmcMThu2EbYL2xmNypq7OT5mQ9vvlFRUS3PL0EgxOCADUwCncQmcYY4HUiAv4dkLaNgFIyCUTC8AQCWSkWFJW34pwAAAABJRU5ErkJggg==","orcid":"","institution":"Tokyo University of Agriculture and Technology","correspondingAuthor":true,"prefix":"","firstName":"Tomoaki","middleName":"","lastName":"Murakami","suffix":""}],"badges":[],"createdAt":"2024-01-15 02:44:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3865213/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3865213/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-07983-7","type":"published","date":"2025-07-01T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49994843,"identity":"e442883e-43f9-42b4-b44b-c5ca10f301cd","added_by":"auto","created_at":"2024-01-22 19:38:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21574087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological distribution of heterozygous amyloid A in the kidney. \u003c/strong\u003e(a) Macroscopic image of kidney tissue stained with Congo red. The cortex is shown on the left, and the medulla-papillae on the right. Bar, 500 µm. (b-d) Higher magnification images of the three boxed areas in Figure 1a: the glomeruli (b), outer medulla (c), and papilla (d). The insets show dichroic birefringence of amyloid deposits under polarized light. Bars, 100 µm. (e) All renal amyloid deposits were positive for anti-Pan-SAA antibody. Bar, 500 µm. (d) Glomerular amyloid deposits are positive for anti-SAA\u003csup\u003eQ45\u003c/sup\u003e antibody, but medullary amyloid deposits are negative except in the papilla. Bar, 500 µm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3865213/v1/d200755aed17db0bfe5b89f8.png"},{"id":49994841,"identity":"6b67ac88-ed0c-4fee-a996-e6a28e366d0d","added_by":"auto","created_at":"2024-01-22 19:38:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":373076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePeptides composition of renal amyloid deposits.\u003c/strong\u003e (a) The pairwise alignment of heterozygous SAA in this patient. Three amino acid substation sites are highlighted in blue (Lys29-Gln45-Ser75 in SAA\u003csup\u003eKQS\u003c/sup\u003e) or red (Ile29-Arg45-Asn75 in SAA\u003csup\u003eIRN\u003c/sup\u003e). Vertical bars (|) indicate fully conserved residues. Colon (:) and period (.) indicate conserved residues between groups with strongly or weakly similar properties, respectively. (b-d) Mapping of SAA-derived peptides detected from glomerular (b), outer medullar (c), and papillary (d) amyloid deposits by mass spectrometry. For each region, three and two samples were digested by trypsin (T1-T3) and chymotrypsin (C1-C2), respectively, and submitted to mass spectrometry. Detected peptides unique to SAA\u003csup\u003eKQS\u003c/sup\u003e and SAA\u003csup\u003eIRN\u003c/sup\u003e are highlighted in blue and red, respectively, while peptides common to both are shown in gray. Ile29 is detected in all regions, but Lys29 was absent. For the 45th residue, Gln45 was detected in the glomeruli, Arg45 in the outer medulla, and both in the papilla.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3865213/v1/77875d93d6ce5cdabb433986.png"},{"id":85968803,"identity":"0aec84fc-ee91-4d74-b1bb-01414195d74b","added_by":"auto","created_at":"2025-07-03 17:50:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26327362,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3865213/v1/a4d8037c-44ab-4a3d-8756-f695dd005cbb.pdf"},{"id":49994844,"identity":"8c1e79bb-7e0a-46c2-90ee-977c7daac9b3","added_by":"auto","created_at":"2024-01-22 19:38:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10744722,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials240115.docx","url":"https://assets-eu.researchsquare.com/files/rs-3865213/v1/13bcf474589881719ea5a6eb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Divergent renal localization patterns of heterozygote-derived two distinct AA amyloids in a cat","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSystemic amyloidosis is a group of intractable diseases characterized by amyloid deposition in multiple organs due to misfolding of serum proteins\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among systemic amyloidoses, amyloid A (AA) amyloidosis caused by serum amyloid A (SAA) is one of the most common diseases in humans and animals\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The kidneys are the primary target organ for AA amyloidosis, but the distribution of deposits varies from patient to patient. For example, in cattle, some individuals exhibit a glomerular predominant distribution of amyloid deposition, while others have predominant deposition in the medullary tubulointerstitium\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, the factors responsible for the difference in deposition distribution have not been elucidated in animals. In humans, although glomerular involvement is the most common presentation of AA amyloidosis, some conditions with predominant deposition in vessels or tubules have also been described\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, and these heterogeneous distributions have been explained by conformational differences resulting from different lengths of the SAA fragments that comprise the amyloid\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. On the other hand, the diversity of AA amyloid distribution in the cortex and medulla is largely unexplored in humans because most analyses of renal amyloidosis in humans are based on cortical biopsy. The distribution of amyloid deposition within the kidney is closely related to clinical manifestations, especially glomerular deposition leads to lethal nephrotic syndrome\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, Therefore, it is essential to elucidate what factors direct the amyloid distribution. In this study, we analyzed renal amyloid deposits in a cat expressing two heterozygous SAAs and found that the primary structure of SAAs affects the intrarenal distribution of AA amyloid deposition.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCase Information\u003c/h2\u003e \u003cp\u003eThe patient, a 4-year-old spayed female cat, presented to the authors' (MT and TA) veterinary clinic with the chief complaint of vomiting, loss of energy, and anorexia. The patient had previously received the feline immunodeficiency virus/feline leukemia virus test at another veterinary hospital was negative (test kit unknown). The patient had numerous ulcers in the oral cavity and a mass on the lower part of the tongue, which was treated with antibiotics and anti-inflammatory drugs. The mass in the hypoglossal area was removed under general anesthesia and submitted to a commercial pathology lab, which was histopathologically diagnosed as lymphoplasmacytic stomatitis with granulation tissue.\u003c/p\u003e \u003cp\u003eAbout a year later, at age 5, in addition to the symptoms of gingivostomatitis, the patient began to exhibit polyuria and polydipsia, chronic renal insufficiency, and non-regenerative anemia. Therefore, she was treated with hematopoietic agents, oral medications for renal protection, and subcutaneous fluid replacement. Later, however, she showed gastrointestinal symptoms, then dehydration, further deterioration of renal function, hypothermia, loss of energy, anorexia, and severe non-regenerative anemia. Ultrasonography revealed mixed hypo- and hyperechoic nodules in the enlarged liver, so a fine needle aspiration of the liver was performed. Fine needle aspiration samples were submitted to a commercial pathology lab and cytologically diagnosed as hepatic amyloidosis.\u003c/p\u003e \u003cp\u003eDespite various symptomatic treatments, including two blood transfusions and hematopoietic agents for severe anemia, antibiotics, anti-inflammatory drugs, and hepatoprotective drugs, the patient died 14 days after the cytological diagnosis of amyloidosis.\u003c/p\u003e \u003cp\u003ePostmortem necropsy was performed promptly, and the liver and kidneys were sampled for histological examination. The liver was multinodularly enlarged, and blood clots were observed on the surface (Supplementary Fig.\u0026nbsp;1a). The kidney was slightly faded and had an uneven surface (Supplementary Fig.\u0026nbsp;1b).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eHistologically, severe amyloid deposition was observed in the kidneys and liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In the kidney, amyloid deposits were widely observed in the cortical glomeruli and medullary interstitium, with particularly severe deposits in the outer medulla (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSAA genes in the case\u003c/h2\u003e \u003cp\u003eReverse transcription-polymerase chain reaction (RT-PCR) and sequencing revealed eight heterozygous single nucleotide substitutions (c.111G\u0026thinsp;\u0026gt;\u0026thinsp;C, c.140A\u0026thinsp;\u0026gt;\u0026thinsp;T, c.183T\u0026thinsp;\u0026gt;\u0026thinsp;T, c.183T\u0026thinsp;\u0026gt;\u0026thinsp;C, c.188A\u0026thinsp;\u0026gt;\u0026thinsp;G, c.231T\u0026thinsp;\u0026gt;\u0026thinsp;C, c.252A\u0026thinsp;\u0026gt;\u0026thinsp;G, c.272G\u0026thinsp;\u0026gt;\u0026thinsp;A, c.282T\u0026thinsp;\u0026gt;\u0026thinsp;C) in the \u003cem\u003eSAA\u003c/em\u003e gene resulting in three amino acid substitutions (K29I, Q45R, S75N). Cloning of PCR products revealed that the amino acid substitution combinations are Ile29-Arg45-Asn75 (SAA\u003csup\u003eIRN\u003c/sup\u003e) and Lys29-Gln45-Ser75 (SAA\u003csup\u003eKQS\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). BLAST analysis using the UniProt database showed that SAA\u003csup\u003eKQS\u003c/sup\u003e and SAA\u003csup\u003eIRN\u003c/sup\u003e were identical to entries M3WHE0 and A0A337SKP2, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDistribution of heterozygote-derived amyloids\u003c/h2\u003e \u003cp\u003eTo determine the respective renal distribution of amyloid derived from SAA\u003csup\u003eIRN\u003c/sup\u003e and SAA\u003csup\u003eKQS\u003c/sup\u003e, immunohistochemistry was performed using anti-SAA\u003csup\u003eQ45\u003c/sup\u003e antibody, which specifically recognizes only SAA with Gln45, and anti-Pan-SAA antibody, which recognizes both SAAs with Gln45 or Arg45. All the renal amyloid deposits were positive for the anti-Pan-SAA antibody, indicating AA amyloidosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Immunohistochemistry using anti-SAA\u003csup\u003eQ45\u003c/sup\u003e antibody was positive for amyloid deposits in the glomeruli, but mostly negative for the medullary deposits except in the papillae (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). There were no noticeable distribution differences between the two antibodies in hepatic amyloid deposits (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eMass spectrometry demonstrated that SAA is the major component of amyloid deposits in glomeruli, outer medulla, and papilla (Supplementary Table\u0026nbsp;1). Analysis of SAA-derived peptides detected by mass spectrometry revealed that N-terminal Gln1-Arg72-derived peptides were frequently detected in all three regions, while C-terminal peptides were rare (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). In all three regions, peptides with Ile29 were detected, but no peptides with Lys29 were detected. Focusing on the 45th amino acid residue, only Glu45 was detected in the glomeruli (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), only Arg45 in the outer medulla (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), and both Glu45 and Arg45 were in the papilla (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The partial overlap of the mass spectrometric results of amyloid collected from the papilla with those of the outer medulla is expected because the two were mixed during microdissection. Therefore, the following discussion will primarily focus on the glomerular and outer medullary results.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBased on immunohistochemistry and mass spectrometry, this cat was diagnosed with AA amyloidosis. Genetic analysis showed that this cat had two heterozygous SAAs (SAA\u003csup\u003eKQS\u003c/sup\u003e, SAA\u003csup\u003eIRN\u003c/sup\u003e), and immunohistochemistry and mass spectrometry further revealed that these two SAAs showed different distributions in nephron: SAA\u003csup\u003eKQS\u003c/sup\u003e-derived amyloid in the glomeruli and papilla, while SAA\u003csup\u003eIRN\u003c/sup\u003e-derived amyloid in the medulla. Functionally, the glomerulus is a mass of capillaries and produces primitive urine. Renal tubules in the medulla reabsorb the necessary components from primitive urine. The concentrated urine passes through the collecting duct and is released through the papillae into the pelvis. Since the pH and protein concentration in urine change dizzyingly as it passes through the nephrons, it may be natural that different biochemical properties of the amyloid would change the deposition site. This study has demonstrated that a few differences in the primary structure of amyloid precursor protein affect the intra-nephron distribution of amyloid deposition. This is the first report to demonstrate that two types of amyloid derived from heterozygotes show different distributions in a single individual.\u003c/p\u003e \u003cp\u003eNotably, although the detection of Arg45 and Gln45 in the central region of SAA varied by deposition site, peptides containing Ile29 in the N-terminal region were consistently detected from each area. The protofilament polypeptides that make up the feline AA fibrils have 11 β-strands in an extended hairpin structure, with the Ile29 located on strand β4 at the beginning of the central β-arch; the hydrophobic side chain of Ile29 faces the inside of the β-arch and forms hydrophobic bonds with side chains of Pro48, Trp52, and Ala54\u003csup\u003e6\u003c/sup\u003e. Therefore, substituting the hydrophobic residue isoleucine for the basic residue lysine is expected to significantly affect the conformation around this region. Mass spectrometry analysis suggests that SAAs\u003csup\u003eIRN\u003c/sup\u003e can form amyloid by themself, but the central region of SAA\u003csup\u003eKQS\u003c/sup\u003e may acquire amyloidogenicity by interacting with the N-terminus of SAA\u003csup\u003eIRN\u003c/sup\u003e. The role of Ile29 in feline AA amyloidosis and the interaction between heterozygous SAAs should be investigated in the future.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides evidence that differences in the primary structure of the precursor proteins affect the distribution of amyloid deposition by showing that heterozygote-derived two types of AA amyloid exhibit different renal distributions within a single individual. Different renal distribution of amyloid deposits produces different clinical manifestations; glomerular amyloid deposition causes proteinuria, while deposition in the tubular interstitium causes impaired urine concentration.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Future application of this study may enable the prediction of the AA amyloid-deposited site within the kidney and the prognosis by evaluating the SAA sequence of each patient.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Analysis\u003c/h2\u003e \u003cp\u003ePortions of the liver and kidney were formalin-fixed and paraffin-embedded, cut into 3-\u0026micro;m sections, and stained with hematoxylin and eosin, and Congo red. Amyloid deposits were confirmed as Congo red-positive material showing yellow to green birefringence under polarized light.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSequencing of the SAA genes\u003c/h2\u003e \u003cp\u003eRT-PCR and sequencing were performed to determine the sequence of \u003cem\u003eSAA\u003c/em\u003e. Primers designed using Primer Blast in NCBI based on the mRNA sequence of predicted \u003cem\u003eFelis catus\u003c/em\u003e serum amyloid A (NCBI accession: XM_045038041.1) are as follows: forward, 5\u0026rsquo;-AGCTCTTCTCCACTGGGACT-3'; reverse, 5\u0026rsquo;-CTCTCCTGAGTGCAGAGCAA-3'. The total RNA was extracted from formalin-fixed paraffin-embedded liver tissue sections using the innuPREP FFPE total RNA Kit (Analytik Jena, \u0026Uuml;berlingen, Germany). One-step RT-PCR was performed using the PrimeScript One-Step RT-PCR Kit (Takara Bio, Shiga, Japan). RT-PCR was performed as follows: reverse transcription at 50\u0026deg;C for 30 min, 94\u0026deg;C for 30 s, 60\u0026deg;C for 30 s, 72\u0026deg;C for 1 min cycles 35 times with a final extension at 72\u0026deg;C for 3 min. Product size was analyzed using agarose gel electrophoresis, and target DNA fragments (446 bp) were purified from the gel using NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL, D\u0026uuml;ren, Germany). Nucleotide sequences were determined using the contract service of Eurofins Genomics (Tokyo, Japan). Each amino acid sequence was translated from the sequenced codon using EMBOSS Transeq\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCloning and sequencing of heterozygous SAA\u003c/h2\u003e \u003cp\u003eThe amplified PCR products were cloned using pGEM\u0026reg;-T Easy Vector Systems (Promega, Madison, WI, USA) and sent for a sequencing service (Greiner Bio-One, Frickenhausen, Germany). Amino acid sequences were translated from the sequenced codon using EMBOSS Transeq\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The determined SAA sequences were compared to the SAA sequences registered in UniProt by BLAST\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eA new antibody was generated for DKYFHARGNYDAAQRGPGG (corresponding to residues 32\u0026ndash;50 of mature cat SAA), a sequence of SAA amino acid sequence that is well conserved in animals\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, using the contract service of Cosmo Bio Co. (Tokyo, Japan). Target sequence synthetic peptide [C\u0026thinsp;+\u0026thinsp;DKYFHARGNYDAAQRGPGG] was immunized to rabbits, and their serum was obtained. In addition to the newly produced SAA antibody, a commercially available SAA antibody (PAA885Hu01, Cloud-Clone Corp., USA) was used as the primary antibody. The newly produced SAA antibody and the commercial SAA antibody are referred to as anti-SAAQ45 antibody and anti-Pan-SAA antibody, respectively, based on the verification of cross-reactivity described in Supplementary Methods and Supplementary Fig.\u0026nbsp;3. Horseradish peroxidase-conjugated polymer anti-rabbit IgG antibody (Dako, Santa Clara, CA, USA) was used as the secondary antibody, and a positive reaction was obtained with 3,3'-diaminobenzidine tetrahydrochloride. Normal rabbit serum was used instead of the primary antibody for the negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTissue Microdissection and Mass Spectrometry\u003c/h2\u003e \u003cp\u003eLiquid chromatography-tandem mass spectrometry (LC-MS/MS) of amyloid deposits collected from Congo red-stained sections was performed to determine the protein profile in the amyloid deposits. As in the previous study\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, Congo red-positive amyloid deposits in glomeruli, outer medulla, and papillae were collected from five regions of approximately 500,000 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e each under a stereomicroscope. Three samples of each collection were digested with trypsin, and two samples were digested with chymotrypsin before LC-MS/MS analysis, as described previously\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFirst, to clarify the protein composition in amyloid deposits, the MS/MS data of three tryptic samples were collated to theoretical fragment patterns of tryptic peptide sequences from the UniProt database using Mascot Server (Matrix Science Inc). The \u003cem\u003ein silico\u003c/em\u003e proteolytic enzymes \"Trypsin/P\" in the Mascot Server's drop-down list were selected. Statistically significant proteins/peptides were extracted by Mascot's probability-based scoring algorithm.\u003c/p\u003e \u003cp\u003eNext, MS/MS data were collated to two SAA sequences (SAA\u003csup\u003eKQS\u003c/sup\u003e and SAA\u003csup\u003eIRN\u003c/sup\u003e) identified in this study to determine which of the heterozygous SAAs the amyloid deposited in each region derived from. The \u003cem\u003ein silico\u003c/em\u003e enzymes \"semiTrypsin\" and \"none\" in the Mascot Server's drop-down list were selected to identify nontryptic and nonchymotryptic peptides, respectively. Statistically significant peptides were extracted using Mascot's probability-based scoring algorithm. The distribution of detected peptides was mapped in each SAA sequence.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThis research was supported by JSPS KAKENHI (Grant No. 23H02380) and the Program on Open Innovation Platform with Enterprises, Research Institute, and Academia (OPERA) from the JST.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eNK and TM conceptualized the paper. NK, MK, SI, YI, and MH performed the experiments and validated the study. NK, MK, and TM performed the formal analysis. NK, SI, YK, and NSM investigated. MT and TA recorded clinical matters and provided resources. NK and TM wrote the original draft and visualized the study. All authors reviewed and TM edited. MK, YI, and TM supervised. TM performed project administration and acquired funding.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAll data are available in the main text or the Supplementary Materials. Please contact the corresponding author ([email protected]) for general comments or material requests. The data supporting the findings of this study can be provided upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAll the authors declared no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBuxbaum, J. N. \u003cem\u003eet al.\u003c/em\u003e Amyloid nomenclature 2022: update, novel proteins, and recommendations by the International Society of Amyloidosis (ISA) Nomenclature Committee. \u003cem\u003eAmyloid\u003c/em\u003e 29, 213\u0026ndash;219 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinney, J. H. \u0026amp; Lachmann, H. J. Systemic AA Amyloidosis. in \u003cem\u003eProtein Aggregation and Fibrillogenesis in Cerebral and Systemic Amyloid Disease\u003c/em\u003e (ed. Harris, J. R.) vol. 65 541\u0026ndash;564 (Springer Netherlands, 2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaram, S., Haidous, M., Royal, V. \u0026amp; Leung, N. Renal AA amyloidosis: presentation, diagnosis, and current therapeutic options: a review. Kidney International 103, 473\u0026ndash;484 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurakami, T. \u003cem\u003eet al.\u003c/em\u003e Atypical AA amyloid deposits in bovine AA amyloidosis. Amyloid 19, 15\u0026ndash;20 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanerjee, S. \u003cem\u003eet al.\u003c/em\u003e Amyloid fibril structure from the vascular variant of systemic AA amyloidosis. Nat Commun 13, 7261 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchulte, T. \u003cem\u003eet al.\u003c/em\u003e Cryo-EM structure of ex vivo fibrils associated with extreme AA amyloidosis prevalence in a cat shelter. Nat Commun 13, 7041 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalighi, M. A., Dean Wallace, W. \u0026amp; Palma-Diaz, M. F. Amyloid nephropathy. Clinical Kidney Journal 7, 97\u0026ndash;106 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadeira, F. \u003cem\u003eet al.\u003c/em\u003e Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res 50, W276\u0026ndash;W279 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThe UniProt Consortium. UniProt: the Universal Protein Knowledgebase in 2023. Nucleic Acids Research 51, D523\u0026ndash;D531 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu, J., Yu, Y., Zhu, I., Cheng, Y. \u0026amp; Sun, P. D. Structural mechanism of serum amyloid A-mediated inflammatory amyloidosis. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e 111, 5189\u0026ndash;5194 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurakami, T. \u003cem\u003eet al.\u003c/em\u003e Identification of novel amyloidosis in dogs: α-S1-casein acquires amyloidogenicity in mammary tumor by overexpression and N-terminal truncation. Vet Pathol 60, 203\u0026ndash;213 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIwaide, S. \u003cem\u003eet al.\u003c/em\u003e Fibrinogen Aα-chain amyloidosis outbreaks in Japanese squirrels (Sciurus lis): a potential disease model. The Journal of Pathology 261, 96\u0026ndash;104 (2023).\u003c/span\u003e\u003c/li\u003e\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":"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":"","lastPublishedDoi":"10.21203/rs.3.rs-3865213/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3865213/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmyloid A (AA) amyloidosis poses a fatal threat to both humans and animals. While the kidneys represent the principal organ affected in AA amyloidosis, there exists variability in the localization of amyloid deposition, with distinct symptoms delineated by the specific deposition sites. Nevertheless, the factors contributing to the diversity of deposition remain unclear. In this study, we identified an association between serum amyloid A (SAA) polymorphisms and patterns of amyloid deposition. Histopathological analysis of the kidneys from a 5-year-old spayed female Japanese cat, which succumbed to systemic AA amyloidosis, revealed renal amyloid deposition in cortical glomeruli and medullary interstitium. Genetic analysis disclosed that the afflicted cat possessed a heterozygous SAA with three amino acid substitutions (K47I, Q63R, S93N), resulting in the SAA\u003csup\u003eKQS\u003c/sup\u003e and SAA\u003csup\u003eIRN\u003c/sup\u003e variants. Mass spectrometry and immunohistochemistry demonstrated that SAA\u003csup\u003eKQS\u003c/sup\u003e was deposited in the glomerulus and renal papilla, while SAA\u003csup\u003eIRN\u003c/sup\u003e was restricted to the extramedullary zone. This study established the differing renal distributions of two AA amyloid variants originating from heterozygotes within a single individual. The evidence supports the notion that the primary structure of precursor proteins defines the distribution of amyloid deposition.\u003c/p\u003e","manuscriptTitle":"Divergent renal localization patterns of heterozygote-derived two distinct AA amyloids in a cat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-22 19:38:08","doi":"10.21203/rs.3.rs-3865213/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":"f7d8d357-599a-4658-8fbd-fe4703e367d2","owner":[],"postedDate":"January 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28260028,"name":"Health sciences/Pathogenesis"},{"id":28260029,"name":"Health sciences/Nephrology/Kidney diseases"},{"id":28260030,"name":"Biological sciences/Molecular biology/Protein folding/Protein aggregation"},{"id":28260031,"name":"Biological sciences/Biochemistry/Peptides"}],"tags":[],"updatedAt":"2025-07-03T17:50:16+00:00","versionOfRecord":{"articleIdentity":"rs-3865213","link":"https://doi.org/10.1038/s41598-025-07983-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-01 00:00:00","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2024-01-22 19:38:08","video":"","vorDoi":"10.1038/s41598-025-07983-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-07983-7","workflowStages":[]},"version":"v1","identity":"rs-3865213","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3865213","identity":"rs-3865213","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}