Fetuin a for the treatment of kidney disorders

Abstract

The present invention relates to fetuin A (AHSG) for use in a method for treating a renal condition, wherein an amount of fetuin A effective for treating said renal condition is administered to a subject in need thereof. The present invention further relates to a pharmaceutical composition for use in treating a renal condition, comprising fetuin A and optionally at least one pharmaceutically acceptable carrier.

Description

[0001] This invention relates to fetoglobulin A (AHSG) for use in methods for treating kidney disease, wherein an effective amount of fetoglobulin A for treating said kidney disease is administered to a subject in need, particularly wherein said kidney disease is ischemic. The invention also relates to pharmaceutical compositions for use in treating kidney disease, comprising fetoglobulin A and optionally at least one pharmaceutically acceptable carrier.

[0002] The goal of modern medicine is to provide new treatments, including novel targets, to achieve better outcomes for specific patient populations. Treatments that take into account patient needs or risks may rely on discoveries, such as targeting pathological signaling cascades or dysfunctional cellular responses. Pharmaceutical protein chemistry is a broad field that uses physics, chemistry, biology, bioinformatics, and medical techniques to analyze and characterize protein structures and mechanisms, identify fundamental molecular and genetic errors in diseases, and develop interventions to correct them. This perspective emphasizes cellular and / or molecular phenomena and implications, rather than the previous conceptual and observational focus on patients and their organs. Protein-based therapies require appropriate proteins and treatment recommendations to identify patients who will benefit from certain treatments. Therefore, the development of treatments critically depends on investigations into new molecularly targeted drugs and procedures.

[0003] Ischemia-reperfusion injury (IRI) often involves a wide range of pathologies, including ischemic stroke, myocardial infarction, acute kidney injury, cardiovascular surgery, and organ transplantation. This reoxygenation, which occurs when blood supply returns to tissues after ischemia, leads to tissue damage. Paradoxically, this essential reoxygenation exacerbates cellular dysfunction and apoptosis after blood flow is restored to previously ischemic tissues. IRI occurs in a wide range of organs, including the heart, lungs, kidneys, intestines, skeletal muscle, and brain, and may involve not only the ischemic organ itself but may also induce systemic damage to distant organs, potentially leading to multi-system organ failure. Reperfusion injury is a multifactorial process leading to widespread tissue destruction. Initially, the lack of sufficient blood flow results in a deficiency of oxygen supply, glucose, and other substances required for normal mitochondrial metabolism, including reduced levels of ATP. This deficiency in ATP production triggers cellular ATP-dependent ion pumps (including Na+) + / K + and Ca 2+The pumps fail, and the transmembrane ion gradient is lost. Damaged or dying cells suffer from calcium overload, characterized by calcium accumulation in mitochondria or apoptotic bodies, respectively. The accompanying low ATP levels (in dysfunctional mitochondria) and pyrophosphate levels (an important inhibitor of calcium calcification) increase the calcification tendency of these organelles. A known complication of organ transplantation (which may also be due to IRI) is delayed graft function (DGF). DGF describes a phenomenon in which recipients who have undergone transplantation have poor graft function and require dialysis during the first few weeks post-transplantation.

[0004] Treatment programs aimed at making organs more resistant to damage caused by ischemia and reperfusion have yielded controversial results or are just emerging. Therapeutic options for enhancing organ resistance to ischemia-reperfusion injury (IRI) or for salvaging organs after sudden oxygen restriction have been the subject of major research efforts over the past decades. Such strategies include, for example, ischemia preconditioning, an experimental strategy in which exposure to a brief, non-fatal ischemic episode results in reduced tissue damage during subsequent IRI; metabolic strategies that increase ischemia tolerance (glycolytic enzymes, erythropoietin, etc., under the control of the transcription factor HIF-1α); therapeutic gases, such as hydrogen, nitric oxide, hydrogen sulfide, and carbon monoxide; and microRNAs (miRNAs) as therapeutic targets. Unfortunately, these approaches have yielded controversial results or are just emerging. Therefore, the treatment of kidney diseases (including, but not limited to, IRI) remains a significant unmet need.

[0005] Therefore, there is a need to provide reagents for treating kidney diseases, and the present invention provides a direct method to address kidney diseases in various scenarios, including ischemia-reperfusion injury.

[0006] Naturally occurring fetoglobulin A (also known as α-2-HS-glycoprotein (AHSG)) is a liver-derived plasma glycoprotein encoded by the AHSG gene. Fetoglobulin A has been described in the art for attenuating early ischemic brain injury in brain scenarios, where inflammation driven by IRI has been reduced in rats with ischemic brain tissue (see, e.g., Wang et al., J Cereb Blood Flow Metab. 2010, 30(3):493-504). Fetoglobulin A has further been associated with the prevention of brain damage in stroke (WO 00 / 60943). However, elevated plasma fetoglobulin A levels have also been found to increase the risk of myocardial infarction and ischemic stroke, suggesting that the role of fetoglobulin A as a preventative agent against ischemic brain injury is not fully understood (Weickert et al., Circulation. 2008, 118:2555-2562).

[0007] Conflicting data have emerged regarding the use of fetoglobulin A as a biomarker for kidney disease. For example, recent studies (exploring the role of fetoglobulin A as a biomarker in kidney transplantation) have failed to provide predictive information on patient clinical outcomes (see, for example, Roos et al., Kidney Blood Press Res. 2011, 34(5):328-33; Mersai et al., Urol J. 2015, 12(3):2182-6; Koca et al., Nephrology Dialysis Transplantation. 2017, 32(3):iii419-iii420). In these publications, fetoglobulin A levels are typically measured in peripheral blood, which does not reflect fetoglobulin A levels in tissues. In summary, these publications suggest that fetoglobulin A levels in the blood are not a useful biomarker for long-term survival or clinical prognosis in patients with kidney disease.

[0008] However, in the context of this invention, it was surprisingly found that the administration of fetoglobulin A reduced kidney tissue damage induced by hypoxia in the kidneys.

[0009] Therefore, in a first aspect, the present invention relates to fetoglobulin A (AHSG) for use in a method for treating kidney disease, wherein an effective amount of fetoglobulin A for treating said kidney disease is administered to a subject in need. Preferably, said kidney disease is caused by hypoxia, such as hypoxia during transplantation, cardiovascular surgery, or other major surgery. The kidney disease may be ischemic kidney disease.

[0010] Unbound by any theory and based on the experimental evidence presented in this paper, it is believed that when fetoglobulin A is administered to subjects in need, particularly those with ischemic nephropathy, it acts as a “mineral scavenger” in hypoxic kidney tissue by “clearing” calcium levels. Fetoglobulin A is thought to counteract ectopic calcification, thereby maintaining kidney tissue integrity and preventing downstream inflammatory cascades that may lead to renal fibrosis through IRI-induced collagen expression. The explanation presented in this paper is that fetoglobulin A acts as a “mineral molecular chaperone” in nephropathy, protecting the organ from ischemia-reperfusion injury in the kidney by clearing calcium-containing particles and mitigating harmful inflammatory responses.

[0011] As used herein, “renal condition” is defined as one or more conditions or diseases affecting the kidneys. The term encompasses, but is not limited to, kidney diseases. For example, kidney injury not caused by disease may also be a renal condition (e.g., some injuries resulting during major surgery; although major surgery can also be a cause of kidney disease). Preferably, the renal condition is ischemic renal condition, i.e., a kidney condition caused by and / or associated with ischemia. When a renal condition “is” a condition, this does not preclude the presence of other conditions, and multiple other conditions may occur independently or concurrently and interdependently with the stated renal condition; for example, IRI and DGF may occur simultaneously.

[0012] In a preferred embodiment of the invention, the kidney condition is selected from the group consisting of: acute kidney condition, chronic kidney condition, renal fibrosis, chronic kidney disease (CKD), renal insufficiency, renal inflammation, acute kidney injury (AKI), ischemic kidney condition, condition related to renal hypoxia, renal ischemia-reperfusion injury (IRI), kidney tissue damage, kidney transplant-related condition, cardiovascular surgery-related condition, and combinations thereof, particularly wherein the kidney tissue damage is ischemic kidney tissue damage. Cardiovascular surgery-related conditions can be, for example, kidney tissue damage caused by cardiovascular surgery (particularly, by hypoxia and / or ischemia during cardiovascular surgery).

[0013] In another embodiment of the invention, the ischemic nephropathy is selected from the group consisting of: acute ischemic nephropathy, chronic ischemic nephropathy, renal fibrosis due to ischemia, renal inflammation due to ischemia, acute kidney injury due to ischemia, ischemic nephropathy, conditions related to renal hypoxia, renal ischemia-reperfusion injury, ischemic kidney tissue damage, conditions related to ischemia during kidney transplantation (e.g., delayed graft function), and combinations thereof.

[0014] As used in this article, "acute kidney disease" refers to a sudden loss or decline in kidney function. This can be triggered by acute accidental kidney injury or an acute onset of illness. The causes of this condition are numerous, including but not limited to prerenal, postrenal, and intrinsic acute kidney failure. Systemic diseases (e.g., manifestations of autoimmune diseases), crush injuries, contrast agents, and certain antibiotics can also trigger acute kidney failure.

[0015] "Chronic kidney disease," such as chronic kidney disease (CKD), chronic renal insufficiency, or chronic kidney inflammation such as chronic pyelonephritis, is also included in the possible treatment spectrum, but is not limited to those. "Chronic kidney disease" can be triggered by anatomical localization, including but not limited to: vascular diseases, which include large vessel diseases such as bilateral renal artery stenosis and small vessel diseases such as ischemic nephropathy, hemolytic uremic syndrome, and vasculitis; glomerular diseases; tubulointerstitial diseases; obstructive nephropathy; or congenital diseases such as polycystic kidney disease or Central American nephropathy.

[0016] "Fibrosis" involves the formation of excessive fibrous connective tissue, such as in the kidneys or kidney tissue. Tissue inflammation can trigger fibrosis. In some embodiments, the fibrosis involves interstitial fibrosis.

[0017] As used herein, “ischemia” or “ischemic (of)” means an insufficient blood supply to an organ or specific part of the body. It is generally considered a serious clinical problem. Ischemia results in a reduced blood supply, thus leading to a reduced supply of oxygen and nutrients, and also involves a reduced removal of waste products from tissues. This can lead to irreversible tissue damage and cell death. Ischemia can occur in a variety of kidney disease scenarios and can be the cause of many kidney diseases. Ischemia can also occur in the context of major surgery, for example, it may be caused by major surgery. “Reperfusion” is defined herein as the restoration of blood supply after an ischemic event. It can cause tissue damage by causing an imbalance of minerals, particularly by calcium accumulation, for example, in the kidneys. “Ischemia-reperfusion injury” (IRI), sometimes also called reperfusion injury (RI) or reoxygenation injury, is tissue damage that occurs when blood supply returns to tissues (reperfusion) after a period of ischemia and / or after a period of reduced oxygen supply (hypoxia).

[0018] As used herein, “cardiovascular surgery” refers to surgical treatment of large blood vessels and organs within the chest or abdomen, such as surgery involving the heart, major blood vessels, or lungs. In some implementations, cardiovascular surgery may be coronary artery bypass surgery or abdominal aortic aneurysm surgery. Abdominal aortic aneurysm surgery can cause renal ischemia-reperfusion injury. Kidney disease may be associated with cardiovascular surgery even if it is not performed directly on the kidney. Ischemia can occur during cardiovascular surgery and cause damage to kidney tissue. The development of acute kidney injury (AKI) following cardiovascular surgery is well known, and some implementations involve the treatment of acute kidney injury resulting from cardiovascular surgery.

[0019] In the context of this invention, "subject with this need" generally refers to a person who is expected to have or is at risk of developing kidney disease, or a person who already has kidney disease (particularly has been diagnosed with kidney disease, preferably ischemic kidney disease). The subject may also be referred to as a "patient." The subject may require preventative or therapeutic treatment. In some embodiments, the anticipated increased risk of developing kidney disease may be due to a scheduled major surgery during which ischemia is risky or even anticipated, for example, an increased risk of kidney disease, given a scheduled kidney transplant or cardiovascular surgery. In some embodiments, the subject with this need is expected to undergo or has already undergone major surgery, preferably a kidney transplant or cardiovascular surgery.

[0020] As used herein, “treatment” encompasses both therapeutic treatment and preventative treatment (prevention) of a condition. “Treatment” may include the administration of proteins or compounds described herein to (i) reduce or eliminate at least one symptom of a particular condition, or (ii) slow the progression of the condition, or (iii) stop or inhibit the development of at least one symptom of a particular condition. As used herein, “therapeutic treatment” refers to treatment of an existing condition, i.e., treatment after the onset of said condition, and its purpose is to eliminate, alleviate, or slow the existing condition or reduce its severity. Conversely, as used herein, “preventative treatment” or “prevention” refers to a type of treatment with respect to a condition designed to reduce the risk of developing it or its symptoms and / or signs in a subject who does not currently have said condition, particularly after events such as major surgeries such as transplantation. In a preferred embodiment, therapeutic treatment means improving the prognosis of the condition. In some embodiments, preventative treatment is administered before, during, and / or after major surgeries such as kidney transplantation or cardiovascular surgery to reduce the risk of onset of diseases associated with major surgery (particularly those associated with ischemia during kidney transplantation or cardiovascular surgery). In some further embodiments, the term "treatment" preferably means not only the relief of symptoms of the condition, but also the improvement, delay, or prevention of the condition itself, particularly a reduction in severity or prevention of the condition. Further, "treatment" can include administering or applying a therapeutic agent to a subject or patient, or performing a procedure or program on a subject to obtain a therapeutic or preventive benefit related to the condition. For example, treatment can include administering a pharmaceutically effective amount of fetoglobulin A. In some embodiments, the condition mentioned above is kidney disease.

[0021] In one embodiment, the present invention relates to fetoglobulin A for use in a method for treating kidney disease, particularly therapeutically treating kidney disease, wherein an effective amount of fetoglobulin A for treating said kidney disease is administered to a subject suffering from said kidney disease. In some embodiments, said kidney disease is ischemic kidney disease. In this context, "treatment" means treatment of established kidney disease and / or treatment following the onset of said kidney disease.

[0022] In an alternative embodiment, the invention relates to fetoglobulin A for use in a method for preventing (also known as prophylactic treatment) kidney disease, wherein an amount of fetoglobulin A effective for preventing said kidney disease is administered to a subject in need, particularly a subject at risk of developing the disease in the future, such as a subject scheduled for major surgery that may involve renal ischemia. In the alternative embodiment, "treatment" means "prevention" of kidney disease before the onset of said disease. In some cases, at least one dose of fetoglobulin A is administered within 48 hours prior to surgery involving ischemia (particularly transplant surgery).

[0023] In another embodiment of the invention, the kidney disease is a condition related to kidney transplantation, particularly a condition caused by kidney transplantation, specifically selected from the group consisting of: delayed graft function (DGF), organ rejection of the kidney transplant, kidney tissue damage due to kidney transplantation, inflammation due to kidney transplantation, ischemia-reperfusion injury (IRI) due to kidney transplantation, and combinations thereof. In some embodiments of the invention, the kidney disease is a condition caused by kidney transplantation, specifically selected from the group consisting of: delayed graft function, kidney tissue damage due to kidney transplantation, ischemia-reperfusion injury due to kidney transplantation, and combinations thereof.

[0024] In some implementations, the kidney disease is ischemic kidney disease, particularly ischemic kidney disease resulting from kidney transplantation or cardiovascular surgery.

[0025] In another preferred embodiment of the invention, the kidney disease, particularly ischemic kidney disease, is associated with or caused by hypoxia, especially wherein the hypoxia is caused by ischemia during major surgery (e.g., ischemia during cardiovascular surgery or kidney transplantation).

[0026] As used herein, “major surgery” is defined by invasive surgical procedures, particularly those involving resection. Major surgery can include access to body cavities, removal of organs, organ transplantation, and / or alteration of normal anatomy. In some embodiments, major surgery involves local or general anesthesia of the subject. Generally, a surgery is considered “major surgery” if the mesenchymal barrier is opened (pleural cavity, peritoneum, meninges). For example, kidney transplantation and cardiovascular surgery are major surgeries. This also includes embodiments of “major surgery” according to the present invention when the term “surgery” is used without specifying that it is a major surgery.

[0027] In the context of this invention, "kidney tissue damage" includes all forms of tissue damage, such as incidental damage caused by all hypoxia conditions (especially hypoxia during kidney transplantation, cardiovascular surgery, or other major surgeries).

[0028] As used herein, “hypoxia” refers to a condition in which the body or a region of the body lacks an adequate supply of oxygen at the tissue level. It is often part of a pathological condition. Oxygen-induced tissue damage includes all such conditions in which the oxygen supply is lost or reduced, resulting in insufficient oxygen supply in any tissue. This also includes reduced blood supply, often due to major surgery or damage to blood vessels. The term “hypoxia” also includes hypoxia as an extreme form of low oxygen, namely, complete depletion of oxygen levels.

[0029] In another preferred embodiment of the invention, the IRI is caused by hypoxia during surgical procedures, particularly major surgeries. Hypoxic conditions occurring during such surgeries can lead to ischemia-reperfusion injury in the kidneys, thereby damaging the tissue. This ischemia-reperfusion injury may be caused at least in part by calcification.

[0030] In a further embodiment of the invention, the kidney condition is delayed graft function, renal fibrosis, or a combination thereof.

[0031] As used herein, "fetoprotein A" refers to a protein or fragment thereof or a derivative thereof, whether naturally occurring or non-natural, having the same or similar functions as naturally occurring fetoprotein A, particularly as naturally occurring human fetoprotein A. Preferably, it is a calcification inhibitor and / or has a protein sequence at least partially identical to human fetoprotein A. In some embodiments, fetoprotein A is plasma-derived or recombinant human fetoprotein A. Human plasma-derived fetoprotein A is obtained from human plasma. The term "fetoprotein A" should also encompass all naturally occurring alleles, splice variants, and isotypes, as well as synthetic peptides, peptide mimics, or peptide fragments that have the same or similar functions as naturally occurring and / or human fetoprotein A, such as inhibiting calcification.

[0032] In some embodiments of the invention, the fetoglobulin A is human fetoglobulin A. In some embodiments, the human fetoglobulin A is derived from human plasma, or alternatively, the human fetoglobulin A is recombinant human fetoglobulin A. Plasma-derived human fetoglobulin A can be purified from human plasma. Recombinant human fetoglobulin A can have a different glycosylation pattern than plasma-derived human fetoglobulin A. In some embodiments, the fetoglobulin A is naturally occurring fetoglobulin A. The term "naturally occurring protein" as used in the context of this invention generally refers to a protein that is naturally found in any kind of living organism and can be isolated from the tissues, fluids, and / or individual cells of any kind of organism. For example, some naturally occurring proteins can be obtained from human plasma. Recombinant proteins also include fetoglobulin A derivatives that are labeled or tagged in some way, such as by attaching genetic tags (e.g., FLAG-tag, His-tag, Myc-tag, HA-tag, or fluorescently labeled proteins, such as GFP, YFP, or RFP), which may or may not facilitate purification.

[0033] The term "purified" in this document generally describes protein purification, as is well known to those skilled in the art. Typically, proteins can be purified based on their characteristics such as solubility, size, charge, and specific binding affinity. That is, proteins are often separated based on their net charge using ion-exchange chromatography. Protein purification techniques are well known to those skilled in the art. At one level, these techniques involve homogenizing and fractionating cells, tissues, or organs into peptide and non-peptide fractions. Unless otherwise stated, the target protein or peptide can be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity). Methods particularly suitable for preparing pure peptides include ion-exchange chromatography, gel size exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, hydrophobic interaction chromatography, immunoaffinity chromatography, and isoelectric focusing. One method for purifying peptides is high-performance liquid chromatography (HPLC). Another method for protein purification is rapid protein liquid chromatography (HPLC). For example, the purification of plasma proteins can be performed by plasma fractionation and / or chromatographic steps. Protein purification may also include virus inactivation as a step. The term "purified" does not exclude the presence of some impurities, but generally, other blood components and / or impurities have been largely removed.

[0034] In another preferred embodiment of the invention, a functionally active derivative or fragment of human fetoglobulin A is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or most preferably at least 99% identical and / or homologous to SEQ ID NO:1 as defined below. “Homologous” is defined herein as sequence similarity within different biological sequences at the RNA, DNA, and protein sequence levels. A “functionally active derivative” has the same or similar activity as naturally occurring fetoglobulin A, particularly the ability to inhibit calcification. The percentage of sequence identity can be determined, for example, by sequence alignment. Sequence alignment methods used for comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990, J.Mol.Biol, 215:3, 403-410) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0035] Therefore, in another embodiment of the invention, the fetoglobulin A comprises or is composed of the following sequence: the amino acid sequence according to SEQ ID NO:1 or a fragment thereof. In the context of this invention, the term "fragment thereof" refers to any amino acid sequence or peptide as defined by SEQ ID NO:1, including but not limited to each smaller fragment exhibiting the activity of fetoglobulin A or any fragment containing additional amino acids, as defined above for fragments and derivatives of naturally occurring proteins having the activity of fetoglobulin A. In some embodiments, the length of the fragment is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the length of SEQ ID NO:1, wherein the length relates to the number of amino acids relative to the full length of SEQ ID NO:1. The fragment may contain additional amino acids that are not part of SEQ ID NO:1. Fragments of the proteins of the present invention can be obtained by sequence alterations in said proteins (e.g., by deletion, substitution, and / or addition of one or more amino acids), wherein the protein with the sequence alteration retains the function of the unchanged protein, i.e., its ability to localize to subcellular structures selected from the group consisting of the cell cortex, contractile loop, and intermediates during cell cycle progression, or to function as a fluorescent reporter protein. Such sequence alterations may include, but are not limited to, conserved substitutions, deletions, mutations, and / or insertions.

[0036] The human fetal globulin A sequence (protein and nucleic acid sequences) is available from the National Center for Biotechnology Information (NCBI).

[0037] SEQ ID NO:1 (Protein sequence of human fetal globulin A)

[0038] MKSLVLLLCLAQLWGCHSAPHGPGLIYRQPNCDDPETEEAALVAIDYINQNLPWGYKHTLNQIDEVKVWPQQPSGELFEIEIDTLETTCHVLDPTPVARCSVRQLKEHAVEGDCDFQLLKLDGKFSVVYAKCDSSPADSAEDVRKVCQDCPLLAPLNDTRVVHAAKAALAAFNAQNNGSNFQLE EISRAQLVPLPPSTYVEFTVSGTDCVAKEATEAAKCNLLAEKQYGFCKATLSEKLGGAEVAVTCMVFQTQPVSSQPQPEGANEAVPPTPVVDPDAPPSPPLGAPGLPPAGSPPDSHVLLAAPPGHQLHRAHYDLRHTFMGVVSLGSPSGEVSHPRKTRTVVQPSVGAAAGPVVPPCPGRIRHFKV

[0039] Protein sequence: NP_001341500.1 (368 amino acids)

[0040] (https: / / www.ncbi.nlm.nih.gov / protein / NP_001341500.1)

[0041] SEQ ID NO:2 (Nucleic acid sequence of human fetal globulin A)

[0042]

[0043] Nucleotide sequence: NM_001354571.2 (1107 nucleotides)

[0044] (https: / / www.ncbi.nlm.nih.gov / gene / 197; NM_001354571.2; Common coding sequence CCDS87176.1)

[0045] In another preferred embodiment, a functionally active derivative or fragment of the naturally occurring protein fetoglobulin A is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, or most preferably at least 99% identical and / or homologous to SEQ ID NO:3 (bovine fetoglobulin A sequence) as defined below.

[0046] SEQ ID NO:3 (Protein sequence of bovine placental protein A)

[0047] MKSFVLLFCLAQLWGCHSIPLDPVAGYKEPACDDPDTEQAALAAVDYINKHLPRGYKHTLNQIDSVKVWPRRPTGEVYDIEIDTLETTCHVLDPTPLANCSVRQQTQHAVEGDCDIHVLKQDGQFSVLFTKCDSSPDSAEDVRKLCPDCPLLAPLNDSRVVHAVEVALATFNAESNGSY LQLVEISRAQFVPLPVSVSVEFAVAATDCIAKEVVDPTKCNLLAEKQYGFCKGSVIQKALGGEDVRVTCTLFQTQPVIPQPQPDGAEAEAPSAVPDAAGPTPSAAGPPVASVVVGPSVVAVPLPLHRAHYDLRHTFSGVASVESSSGEAFHVGKTPIVGQPSIPGGPVRLCPGRIRYFKI

[0048] Protein sequence: NP_776409.1 (359 amino acids)

[0049] (https: / / www.ncbi.nlm.nih.gov / protein / NP_776409.1)

[0050] SEQ ID NO:4 (Nucleic acid sequence of bovine fetal globulin A)

[0051]

[0052] Nucleotide sequence: NM_173984.3 (encoding nucleotide sequence (1080 nt))

[0053] (https: / / www.ncbi.nlm.nih.gov / nuccore / NM_173984.3)

[0054] In another embodiment of the invention, the fetoglobulin A is recombinantly expressed, preferably in a host cell system that provides post-translational modifications, more preferably glycosylation.

[0055] As used herein, the term "expression" generally means allowing or causing information in a gene or DNA sequence to become expressed. Specifically, in the context of this invention, it means the intracellular production of a protein encoded by the nucleic acid expression construct of this invention by activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequence. That is, a DNA sequence is expressed in or by cells to form an "expression product," such as a protein. Expression of a protein in or by cells can depend on a wide variety of factors, including, but not limited to, the presence or absence of a promoter sequence operatively linked to the DNA sequence encoding the target protein, enhancer and / or silencing sequences, or other DNA sequences controlling the rate of transcription, cell culture conditions applied to the cells carrying the respective DNA sequences (including the culture medium used to culture the cells), and / or the copy number of the DNA sequence introduced into the cells. Successful expression of a fusion protein can be analyzed and / or visualized using standard methods known to those skilled in the art, including but not limited to Western blot analysis, Northern blot analysis, RNase protection assays, quantitative RT-PCR, and methods involving direct or indirect fluorescence readings. "Recombinant" means artificially produced, involving, or representing an organism, cell, protein, or genetic material formed through recombination. The recombinant expression of the desired protein is a standard method in the art, followed by the standard protein purification method described below.

[0056] Post-translational modifications enhance the functional diversity of the proteome through the covalent addition of functional groups or proteins, the proteolytic cleavage of regulatory subunits, or the degradation of the entire protein. These modifications include, in particular, phosphorylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, lipidation, and glycosylation.

[0057] "A host cell that provides post-translational modifications" refers to a host cell that delivers the necessary modification programs, thereby enabling the correct post-translational modifications of mammalian proteins. Systems that provide these requirements are, for example, insect cell expression systems, such as BTI-Tn5B1-4 or SF9 cell lines, or mammalian cell expression systems, such as, but not limited to, HEK 293(T), CHO, or HELA cells.

[0058] Glycosylation is defined as an enzyme-directed, site-specific process that links sugars to form glycans, which in turn attach to proteins, lipids, or other organic molecules. Protein glycosylation, as a post-translational process, is a natural process in cells through which sugars are selectively added to specific protein residues to transfer greater structural stability or function to the native protein structure. Therefore, glycosylation plays a crucial role in the proper functioning of a wide variety of proteins. Glycosylation includes, but is not limited to, C-linked, N-linked, O-linked, or S-linked glycosylation and glycation. Thus, glycation refers to the non-enzymatic attachment of reducing sugars to the nitrogen atoms of proteins.

[0059] As used herein, “injection” refers to parenteral administration and does not include oral or inhalation administration. As used herein, “injection” encompasses intravenous (iv), intra-articular, intramuscular (im), subcutaneous (sc), and intradermal (id), intraperitoneal, intraosseous, intracardiac, intra-articular, and intracavernosal administration. In some advantageous embodiments, injection refers to intravenous (iv) or subcutaneous (sc) administration. The injection may be discontinuous and / or short-duration, for example, using a needle with a single dose of fetoglobulin A (and optionally, a further dose at a later time). Alternatively, the injection may also be an infusion, i.e., a longer, continuous injection. In some embodiments, the injection is administered in less than 15 minutes, particularly less than 10 minutes or less than 5 minutes. Administration by injection also includes systemic administration, such as via extracorporeal circulation (ECC). As used herein, “inhalation” describes the process of inhalation or aspiration, which delivers air, gases, active substances, and aerosols to the lungs. For example, fetoglobulin A may be provided as part of an aerosol for inhalation purposes.

[0060] In some embodiments of the invention, fetoglobulin A is administered to a patient by injection or inhalation. In some preferred embodiments, fetoglobulin A is administered to a patient by injection. In some embodiments, fetoglobulin A is administered to a patient by intravenous (iv), intramuscular (im), or subcutaneous (sc) injection, preferably intravenous (iv).

[0061] In some embodiments, fetoglobulin A is administered to the patient at an amount effective for treating the kidney condition, particularly at a concentration range of 1 mg / kg to 200 mg / kg body weight, particularly via intravenous administration. In some embodiments, fetoglobulin A is administered to the patient at a concentration range of 5 mg / kg to about 100 mg / kg body weight, particularly via intravenous administration. In some embodiments, fetoglobulin A is administered to the patient at a concentration range of 10 mg / kg to about 50 mg / kg body weight, particularly via intravenous administration. In some embodiments, fetoglobulin A is administered to the patient at a concentration range of 10 mg / kg to about 75 mg / kg body weight, particularly via intravenous administration. In some embodiments, fetoglobulin A is administered to the patient at a concentration range of 20 or 30 mg / kg to about 200 mg / kg body weight, particularly via intravenous administration. In some embodiments, fetoglobulin A is administered to the patient at a concentration range of 20 mg / kg to about 50 mg / kg body weight, particularly via intravenous administration. In some implementations, fetoglobulin A is administered to patients at concentrations ranging from 30 mg / kg to approximately 50 mg / kg body weight, particularly via intravenous administration.

[0062] In a further embodiment, fetoglobulin A is administered to the subject in need during major surgery, such as cardiovascular surgery or kidney transplantation. In a further embodiment, fetoglobulin A is administered to the subject in need after major surgery, such as cardiovascular surgery or kidney transplantation. In a further embodiment, fetoglobulin A is administered to the subject in need before major surgery, such as cardiovascular surgery or kidney transplantation. These embodiments can be combined; for example, in some embodiments, fetoglobulin A is administered both before and after major surgery.

[0063] In the context of this invention, fetoglobulin A can be a target of the transcription factor HIF-1α. HIF-1α, also known as hypoxia-inducible factor 1-α, is a subunit of the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1). "Target" is defined herein as a protein that is affected "downstream" in relation to a cell signaling pathway or cascade. In the context of this invention, fetoglobulin A can function downstream of HIF-1α.

[0064] In some embodiments of the invention, the treatment includes modulating the level of calcification in kidney tissue, preferably calcification levels associated with hypoxia. "Calcification" is defined herein as the accumulation of calcium salts in body tissues, particularly in the kidneys, which can lead to permanent damage to said tissues. It can cause an inflammatory response and tissue damage due to the limited oxygen supply to the tissue. In one embodiment, the treatment includes removing calcium mineral deposits from kidney tissue, particularly from transplanted kidney tissue.

[0065] In another embodiment of the invention, at least two doses of fetoglobulin A or at least three doses of fetoglobulin A are administered. In some embodiments, at least four doses of fetoglobulin A are administered. In another embodiment of the invention, the treatment of the subject includes a treatment phase in which at least one dose of fetoglobulin A is administered at least daily or weekly, preferably in the week or month following an ischemic event and / or after surgery (especially major surgery).

[0066] In the context of this invention, the term "ischemic event" refers to any event that causes and / or includes ischemia (particularly including ischemia). An ischemic event can occur when the blood supply to a tissue is restricted during a surgical procedure. For example, during a kidney transplant procedure, the graft is often not adequately perfused with blood. Sudden blood loss during an accident, during which the kidney is not adequately perfused, can also be an ischemic event. In some embodiments, the ischemic event is ischemia during major surgical procedures, particularly kidney transplantation or cardiovascular surgery.

[0067] In another embodiment of the invention, at least one dose of fetoglobulin A is administered before, during, or after an ischemic event and / or surgical procedure (particularly major surgery). In some embodiments, the ischemic event and / or surgical procedure is a kidney transplant or cardiovascular surgery.

[0068] In another embodiment of the invention, at least one dose of fetoglobulin A is administered within 48 hours (particularly within 24 hours) before or after an ischemic event, such as a kidney transplant or cardiovascular surgery. In some embodiments, at least one dose of fetoglobulin A is administered within 48 hours (particularly within 24 hours) after an ischemic event. In another embodiment, at least one dose of fetoglobulin A is administered within 48 hours (particularly within 24 hours) before a major surgical procedure. In another embodiment, at least one dose of fetoglobulin A is administered within 48 hours (particularly within 24 hours) after a major surgical procedure. In some further embodiments, fetoglobulin A is administered during a major surgical procedure. In some embodiments, the major surgical procedure is a kidney transplant or cardiovascular surgery.

[0069] In another embodiment of the invention, at least one first dose of fetoglobulin A is administered within 48 hours (particularly within 24 hours) before the ischemic event, and at least one second dose of fetoglobulin A is administered within 48 hours (particularly within 24 hours) after the ischemic event. In some embodiments, at least two doses of fetoglobulin A are administered within 48 hours (particularly within 24 hours) before the ischemic event. In another embodiment, at least two doses of fetoglobulin A are administered within 48 hours (particularly within 24 hours) before major surgery. In another embodiment, two or three doses of fetoglobulin A are administered within 48 hours (particularly within 24 hours) before major surgery. In some embodiments, at least two doses of fetoglobulin A are administered within 48 hours (particularly within 24 hours) after the ischemic event. In another embodiment, at least two doses of fetoglobulin A are administered within 48 hours (particularly within 24 hours) after major surgery. In another embodiment, two or three doses of fetoglobulin A are administered within 48 hours (particularly within 24 hours) after major surgery.

[0070] In some implementations, fetoglobulin A is administered to the patient at least once a week, particularly at least once every 48 hours, and preferably at least once every 24 hours during the treatment period.

[0071] In some embodiments, fetoglobulin A is administered in separate doses over a total duration of 1 to 100 days, particularly over a total duration of 1 to 50 days. In another embodiment, fetoglobulin A is administered in separate doses over a total duration of 1 to 25 days. In yet another embodiment, fetoglobulin A is administered in separate doses over a total duration of 1 to 10 days. In yet another embodiment, fetoglobulin A is administered in separate doses over a total duration of 2 to 10 days.

[0072] In one embodiment of the invention, the treatment comprises administering fetoglobulin A as part of a pharmaceutical composition to a subject in need. Preferably, the pharmaceutical composition is in liquid form. Preferably, the pharmaceutical composition is in one or more of the embodiments defined below in a second aspect of the invention.

[0073] In a second aspect, the present invention relates to a pharmaceutical composition for use in the treatment of kidney disease, comprising fetoglobulin A (AHSG) and optionally at least one pharmaceutically acceptable carrier. In some embodiments, this second aspect relates to a pharmaceutical composition for use in a method for treating kidney disease, wherein an amount of fetoglobulin A effective for treating said kidney disease is administered to a subject, preferably a patient, in need of such treatment.

[0074] The term "pharmaceutical carrier" as used herein can refer to any substrate used in drug delivery processes to improve the selectivity, efficacy, and / or safety of drug administration.

[0075] The pharmaceutical compositions according to this second aspect can be used for the same treatments and kidney conditions as those described above with respect to fetoglobulin A, and any treatment described above can also be described as embodiments of the pharmaceutical compositions used in such treatments. In a preferred embodiment of this second aspect, the kidney condition is selected from the group consisting of: acute kidney condition, chronic kidney condition, renal fibrosis, chronic kidney disease, renal insufficiency, renal inflammation, acute kidney injury, ischemic kidney condition, hypoxia-related condition, renal ischemia-reperfusion injury, renal tissue damage (preferably ischemic renal tissue damage), kidney transplant-related condition, cardiovascular surgery-related condition, and combinations thereof.

[0076] In some embodiments, the pharmaceutical composition is a liquid. In some embodiments, the pharmaceutical composition comprises water.

[0077] In another embodiment of the second aspect, the kidney transplant-related condition is selected from the group consisting of: delayed graft function, organ rejection of the kidney transplant, kidney tissue damage due to kidney transplantation, inflammation due to kidney transplantation, renal IRI due to kidney transplantation, and combinations thereof.

[0078] Preferably, the kidney condition is caused by hypoxia during surgery (especially major surgery). For example, the hypoxia can be caused by ischemia during kidney transplantation or cardiovascular surgery.

[0079] In another preferred embodiment, the fetoglobulin A of the pharmaceutical composition is defined as described in detail above. The pharmaceutical composition may comprise any of the fetoglobulin A embodiments described above.

[0080] In the context of the pharmaceutical composition of the present invention, fetoglobulin A is preferably human fetoglobulin A, more preferably fetoglobulin A derived from human plasma.

[0081] In an alternative embodiment, the fetoglobulin A of the pharmaceutical composition is recombinantly expressed, preferably in a host cell system that provides post-translational modifications, particularly glycosylation.

[0082] In this scenario, it is also preferred that the fetoglobulin A is at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical and / or homologous to SEQ ID NO:1. It is also preferred that the fetoglobulin A comprises SEQ ID NO:1 or a fragment thereof, or is composed of SEQ ID NO:1 or a fragment thereof.

[0083] In one embodiment, as described in detail above, the pharmaceutical composition is administered to a subject. Preferably, the pharmaceutical composition is formulated for injection or inhalation.

[0084] The accompanying drawings and embodiments are intended to illustrate various embodiments of the invention. Thus, the specific modifications discussed therein should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various equivalents, variations, and modifications can be made without departing from the scope of the invention, and therefore it should be understood that such equivalent embodiments will be included herein.

[0085] Attached Figure

[0086] Figure 1

[0087] Chronic fetal hypoxia induces intrauterine growth restriction in mice: Figure 1A Experimental setup and analysis time points. Figure 1B Average number of pups per litter: Figure 1C Total weight distribution of fetuses in E18.5 (n=49 for each experimental condition; No: normoxic, Hy: hypoxic, Cc: caloric control). Figure 1D The mean number of nephrons per E18.5 kidney was determined by staining for the glomerular marker nephrin. Unpaired t-tests with Welch correction were performed.

[0088] Figure 2

[0089] Hypoxia-induced gene expression in the kidneys: Figure 2A Hierarchical clustering of cDNA microarray data of renal gene expression profiles in E18.5 fetuses under hypoxia, normoxic, and caloric control groups was performed (n=3 for each experimental condition). White areas indicate induction, and areas with vertical shading indicate inhibition (in... Figure 2A (The degree of induction or inhibition is not specified in the text). The location of Ahsg is marked with an arrow. Genes with at least a 1.3-fold reduction in hypoxia compared to the two normoxic controls were clustered; one-way ANOVA; P < 0.05. Figure 2B -E. Relative mRNA values ​​of Ahsg (B), Apoa2 (C), Fgg (D), and Gys2 (E) in E18.5 kidney (circle) or liver (square) samples. Figure 2FPlasma levels of fetoglobulin A in E18.5 fetuses were assessed by ELISA.

[0090] Figure 3

[0091] Fetoglobulin A expression in the proximal tubule of the fetus induced by hypoxia: Figure 3A-H. Fetoglobulin A staining on E18.5 kidney sections from normoxic (AD) or hypoxic (EH), wild-type (A,C,E,G) or Clcn5 KO (B,D,F,H) fetuses. Arrows in (D,H) indicate intraluminal fetoglobulin A staining, resulting from impaired endocytosis of the low molecular weight protein in the PT of Clcn5 KO mice. (IL) Immunofluorescence staining of nephron segment marker proteins and fetoglobulin A shown on E18.5 kidney sections. PT, proximal tubule; TAL, thick ascending limb; DCT, distal convoluted tubule; CD, collecting duct; scale bar, 300 μm (overview image) or 50 μm.

[0092] Figure 4

[0093] In vitro expression of fetoglobulin A activated by hypoxia: Figure 4A Potential HREs from mouse Ahsg are described, which are used to generate luciferase reporter gene constructs (1–8). Mutated HREs are shown. Figure 4B Luciferase activity in NRK cells transfected with the reported construct. The fold change in luciferase light emission between hypoxic and normoxic culture conditions is shown. Each condition was normalized to the empty vector control (pGL3). Dunnett multiple comparison test. Figure 4C -E. Expression of fetoglobulin A in primary proximal tubule cells (pPTC) isolated from four different mice (C), in rat cell line NRK (D), and in human cell line HK-2 (E) (cultured under normoxic or hypoxic conditions).

[0094] Figure 5

[0095] Fetoprotein A deficiency exacerbates CKD progression in hypoxic IUGR kidneys: Figure 5A -B. Decreased renal function in 9-week-old mice as assessed by protein / creatinine ratio and glomerular filtration rate (GFR). Figure 5B The most obvious fetal hypoxia AhsgKO animals showed an additive effect of hypoxia and fetoglobulin A deficiency (n≥5 for each experimental condition). Figure 5C-D Representative images of picrosirius red staining of collagen on kidney sections derived from P14 fetal hypoxia Ahsg KO mice (D) show stronger and more intricate patterns compared to fetal hypoxia wt mice (C). Figure 5E- The relative expression levels of the fibrosis markers Acta2 (E), Col1a1 (F), and Fn1 (G) were significantly enhanced in the kidneys of fetal hypoxic Ahsg KO mice (n≥5 for each experimental condition). Fisher LSD test (AB). Scale bar, 50 μm.

[0096] Figure 6

[0097] Fetoprotein A deficiency promotes the accumulation of calcium mineral particles and macrophages in hypoxic IUGR kidneys: Figure 6A-I Calcium biomineralization was detected by staining E18.5 kidney sections from normoxic wt (A, D, G), hypoxic wt (B, E, H), or hypoxic Ahsg KO embryos (C, F, I) with ATTO 488 fluorescently labeled fetoglobulin A (488-FA). Hypoxic Ahsg KO mice (C) exhibited the strongest staining intensity in PT compared to normoxic and hypoxic wt mice (A, B), indicating increased mineralized matrix turnover. Arrows point to granular staining patterns, reflecting the significant accumulation of 488-FA in the papillae (F) and cortex (I), which are present only in hypoxic Ahsg KO mice. Figure 6J-M Regarding the genotypes and oxygen conditions shown, representative immunofluorescence staining for macrophage markers F4 / 80 (j', k', l', m') and fetoglobulin A (j"', k"', l"', m"') was performed on E18.5 kidney sections, counterstained with DAPI (j"", k"", l"", m"") and 488-FA (j", k", l", m"). Accumulation of macrophages and diffuse and granular 488-FA staining was detected only in hypoxic Ahsg KO mice (M). Figure 6N Quantification of the total area stained with F4 / 80 in each field of view. Analyze multiple sections from at least 3 mice for each experimental condition. Scale bar, 100 μm.

[0098] Figure 7

[0099] Fetoglobulin A attenuated the expression of hypoxia-induced fibrosis markers: Figure 7A Representative Western blots show the induction of fibronectin and α-smooth muscle actin (α-SMA) in primary proximal tubule cells (pPTCs) isolated from wt or Ahsg KO mice under hypoxic culture conditions. Figure 7B -D. Fetoprotein A supplementation attenuated hypoxia-induced expression of fibrosis markers Acta2 (B), Col1a1 (C), and Fn1 (D) in pPTC (n≥5 for each experimental condition). Figure 7E-F. Fetoprotein A alone, but not BSA, has a reducing effect on the expression of fibrosis markers (n≥4 for each experimental condition). Figure 7G A representative Western blot described how fetoglobulin A supplementation reduced the expression of fibronectin and type I collagen (Col1a1) proteins. Figure 7H TGF-β1 treatment and hypoxia have an additive effect on Smad3 phosphorylation. A representative Western blot is shown. Figure 7I Quantification of Smad3 activation as shown in (H). Unpaired t-test with Welch correction (BD, only used to compare normoxic wt and normoxic Ahsg KO samples), Fisher LSD test (I).

[0100] Figure 8

[0101] Hypoxia (or IRI) induces the deposition of calcium-containing microparticles in the kidneys: calcium-containing microparticles were detected using 488-FA after ischemia-reperfusion (IRP). 488-FA staining on the control side (l) and ischemia-reperfusion (m) at 7 days post-surgery showed the presence of calcium biominerals only in IRP kidneys.

[0102] Figure 9

[0103] Fetoprotein A supplementation attenuates the expression of fibrosis markers following ischemia-reperfusion injury: Figure 9A -F. Supplementation with fetoglobulin A reduced the expression of ischemia-induced Col1a1(A), Col3a1(B), and Col6a1(C), but had no effect on the expression of non-collagenous fibrosis markers Acta2(D), Fn1(E), or Vim(F). One-way ANOVA test was performed.

[0104] Figure 10

[0105] In mice experiencing ischemia-reperfusion injury (IRI), prophylactic administration of human fetoglobulin A resulted in less damage and less fibrotic remodeling. Figure 10A -D. The expression levels of early damage markers Kim1(A), Krt18(B), Krt20(C) and Lcn2(D) were reduced in IRI kidneys pretreated with fetoglobulin A compared with IRI kidneys pretreated with PBS. Figure 10E During IRI, compared to the PBS control, the fibrosis remodeling markers Clu (E) and Tgfb1 (F) were also reduced. Figure 10G In IRI kidneys pretreated with fetoglobulin A, the non-collagenous fibrosis marker Vim(G) was reduced compared to IRI kidneys pretreated with PBS. Figure 10H-J. Collagen expression in Col1a1(H), Col3a1(I), and Col6a1(J) following prophylactic treatment with fetoglobulin A was observed. Figure 10K Compared to mice pretreated with PBS, Arg1(K) expression was suppressed in mice pretreated with fetoglobulin A, indicating a reduced need for collagen synthesis. Figure 10L Pretreatment with fetoglobulin A also reduced the expression of chemokines Ccl2 (L), Il-6 (M), and Tnf-α (N) involved in damage signaling and repair, which are typically increased under ischemic conditions. Legend in Figure 10: Hollow circles: Unoperated control kidneys injected with PBS; Hollow triangles: IRI kidneys injected with PBS; Solid black circles: Unoperated control kidneys injected with fetoglobulin A; Solid black triangles: IRI kidneys injected with fetoglobulin A. P-values ​​in Figure 10 indicate ordinary one-way ANOVA with Tukey correction for multiple comparisons (normal font) and paired t-tests (bold font).

[0106] Figure 11

[0107] Exogenous fetoglobulin A was present in the kidney tissue of mice after intravenous administration. Figure 11A Western blot. Lanes A to C: Unoperated control kidneys injected with human fetoglobulin A; Lanes D to F: IRI kidneys injected with human fetoglobulin A; Lanes G to I: Unoperated control kidneys injected with PBS; Lanes J to L: IRI kidneys injected with PBS; Lane M: Empty; Lane N: Human fetoglobulin A protein for injection. Top: Western blot of endogenous mouse fetoglobulin A using an anti-fetoglobulin A antibody (specific to mouse fetoglobulin A) from abcam (ab187051). Middle: Western blot of human fetoglobulin A using an anti-fetoglobulin A antibody (specific to human fetoglobulin A) from Santa Cruz (sc-133146-HRP). Bottom: Rplp0 used as a loading control. Kidney samples from mice injected with human fetoglobulin A protein show strong bands for human fetoglobulin A (lane AF). Mouse fetoglobulin A was not detected in the control sample (lane GL) injected with PBS. Of all samples, the IRI kidney (lane JL) injected with PBS showed the highest abundance of mouse fetoglobulin A. This was confirmed by using a mouse-specific anti-fetoglobulin A antibody. ab187051 Human fetal globulin A was not detected because the human fetal globulin A protein (lane N) did not give a signal. Figure 11BAn ELISA for mouse fetoprotein A was performed using the MFTA00 ELISA kit (specific to mouse fetoprotein A) from bio-techne. Endogenous mouse fetoprotein A was elevated only in the IRI kidneys injected with PBS, compared to all other samples. Figure 11C An ELISA for human fetoglobulin A was performed using the DFTA00 ELISA kit (specific to human fetoglobulin A) from bio-techne. Human fetoglobulin A was undetectable in kidney samples from mice injected with PBS. The IRI kidney injected with fetoglobulin A had higher tissue levels of human fetoglobulin A than the unoperated contralateral (right) kidney. Figure 11D -F. Following intravenous administration, exogenous fetoglobulin A was present in the kidney tissue of mice. Fluorescently labeled fetoglobulin A (Alexa-488 labeled fetoglobulin A) was present in the proximal tubule (PT) at 15, 30, and 60 minutes after intravenous injection. Fine punctate staining was detected in the proximal tubule at 15 minutes after injection of Alexa-488 labeled fetoglobulin A. The intensity of this staining increased at 30 and 60 minutes post-injection. G, glomerulus. Figure 11B Legend for C: Hollow circle: Unoperated control kidney injected with PBS; Hollow triangle: IRI kidney injected with PBS; Solid black circle: Unoperated control kidney injected with fetoprotein A; Solid black triangle: IRI kidney injected with fetoprotein A. Figure 11B The p-values ​​in C indicate the results of a standard one-way ANOVA with Tukey correction, for multiple comparisons (regular font) and paired t-tests (bold font).

[0108] Figure 12

[0109] Time course of human fetal protein A in mouse serum after intravenous administration: Figure 12A Lanes A and B: 1 minute post-injection; Lanes C and D: 1 hour post-injection; Lanes E and F: 1 day post-injection; Lanes G and H: 1 week post-injection; Lanes I and J: Control PBS injection; Lane K: Human fetoglobulin A protein for injection. Western blot of human fetoglobulin A using a human-specific sc-133146-HRP anti-fetoglobulin A antibody from Santa Cruz. At 1 day post-injection, the amount of human fetoglobulin A detected in serum was reduced compared to 1 minute or 1 hour post-injection. At 1 week post-injection, human fetoglobulin A was undetectable. Mouse fetoglobulin A was not detected with this anti-fetoglobulin A antibody sc-133146-HRP because the control animals injected with PBS did not give a signal. Figure 12BStandardized ELISA data using the DFTA00 ELISA kit (specific to human fetal globulin A) from bio-techne showed a decrease in intravenously injected human fetal globulin A in mouse serum samples. Compared to the injection time point (1 minute), the amount of circulating human fetal globulin A decreased to 80% after 1 hour and to 8.5% after 1 day. After 1 week, human fetal globulin A was no longer detectable, similar to mice injected with PBS (black circles). Figure 12C The time course of human fetoglobulin A (HFA) in mouse serum following intravenous administration. Standardized ELISA data showed a decrease in HFA in mouse serum samples after intravenous injection. Compared to the injection time point (1 minute), the amount of circulating HFA decreased to 80% after 1 hour and to 8.5% after 1 day. After 1 week, HFA was no longer detectable. Example

[0110] Chronic fetal hypoxia induces IUGR in mice. To establish a chronic fetal hypoxia model, pre-mated pregnant mice were exposed to 10% oxygen from E14.5 to E18.5. Figure 1A It was observed that hypoxic mothers (Hy) ate less, despite free access to food, and since caloric restriction itself is a known inducer of IUGR5, a second control group was included in this analysis to rule out the possibility that the findings might be due to reduced caloric intake rather than hypoxia. In this caloric control (Cc) group, normoxic mothers were fed an amount of food matched the reduced amount consumed by hypoxic mice. There was no difference in litter size, placental mass, or fetal viability among the three groups, but maternal weight gain was significantly reduced in hypoxic mothers. Figure 1B Importantly, only fetuses from hypoxic mothers showed LBW, meeting the IUGR criteria. Figure 1C Fetuses from the normoxic or Cc groups did not exhibit this characteristic. The kidneys of hypoxic fetuses were smaller, with significantly fewer nephrons compared to the control group. Figure 1D ).

[0111] Hypoxic fetal kidneys adopted the liver gene expression pattern. Next, gene arrays were used to examine genome-wide expression in hypoxic fetal kidneys. Sixty-two induced genes and 28 repressed genes were identified and compared with the two control groups. Figure 2AOf the induced genes, 17 were known to be regulated by hypoxia, including true HIF target genes transferrin, trefoil factor 3, neurotin, α-1-antitrypsin (Serpina1d), and α-1-antichymotrypsin (Serpina3n). Furthermore, over 20% of the induced genes were frequently purified from calciprotein particles (CPPs), including major CPP components such as fetoglobulin A (Ahsg), albumin, Apo-A1, and thrombin (F2). Functional annotation clustering of the induced genes revealed enrichment of secretory plasma proteins (which are normally transcribed only by the liver) in hypoxic kidneys. Validation of the microarray data by RT-qPCR of the selected genes confirmed an induction of over 10-fold in hypoxic fetal kidneys alone, but not in control kidneys. Figure 2B -E). No changes in mRNA levels were found in fetal liver samples.

[0112] Under hypoxic conditions, fetoglobulin A is produced locally in the proximal tubules. Interestingly, the gene with the highest induction was found in 7 out of 10 annotated groups (Ahsg). Ahsg belongs to the cysteine ​​protease superfamily of cysteine ​​protease inhibitors and encodes the negative acute-phase glycoprotein fetoglobulin A, whose main function is involved in mineralized matrix metabolism. Despite its strong induction in hypoxic kidneys, no significant increase in fetal plasma fetoglobulin A levels could be detected. Figure 2FThis implies a specific local (rather than systemic) functional relevance during the developmental challenge of hypoxia. To further address the functional relevance of Ahsg expression in the fetal hypoxic kidney, its precise localization was examined. Figure 3 shows the immunohistochemistry of the proximal tubules (Fig. 3A, B, E, F) or tubular lumen (Fig. 3C, D, G, H) of the fetal kidney. Strong fetoglobulin A staining distinguished cells in the proximal tubules (PT) regardless of oxygen conditions (Fig. 3A, E). This is due to the filtering and uptake of systemic fetoglobulin A into the PT via macroprotein-dependent endocytosis, which masks any fetoglobulin A produced locally in the hypoxic fetal kidney. To selectively visualize kidney-derived fetoglobulin A protein, a genetic approach was employed to block endocytosis into the PT, an alternative to the pharmacological inhibition of macroprotein-dependent endocytosis using His-sRAP (a histidine-tagged soluble receptor-associated protein). Clcn5 knockout (KO) mice exhibited severely impaired endocytosis of low molecular weight proteins in PT, mimicking Dent disease. Normo-Clcn5 KO kidneys lacked prominent fetoglobulin A staining in PT cells (Fig. 3B). Conversely, strong intraluminal signals were detected (Fig. 3D), absent in wild-type (wt) samples (Fig. 3C), highlighting the impaired endocytosis phenotype of Clcn5 KO mice. However, in addition to the luminal signal, hypoxic Clcn5 KO kidneys showed strong fetoglobulin A staining in PT (Fig. 3F, H), providing evidence that the observed cellular fetoglobulin A staining truly originated in PT. Dual immunofluorescence staining for fetoglobulin A and markers of different renal segments (Fig. 3I-L) confirmed that fetoglobulin A was expressed only in the PT of hypoxic fetal kidneys. Integral in situ hybridization confirmed fetoglobulin A mRNA synthesis in hypoxic fetal kidneys, but absent in normoxic kidneys.

[0113] Ahsg possesses a putative HIF binding site that overlaps with an enhancer region. Since fetoglobulin A has been shown to be produced locally in hypoxic fetal kidneys, the direct activation of Ahsg expression by hypoxia was assessed. To examine potential HIF binding sites (hypoxia response elements - HREs) at the human AHSG locus, HIF-1α (HIF-1-α) and HIF-2α (HIF-2-α) ChIP-seq datasets derived from hypoxic MCF7 cells were used. Clusters of potential HREs near exon 4 of human AHSG were identified, overlapping with H3K27Ac and H3K4Me1 (chromatin markers of active enhancer elements) and DNase I hypersensitivity. Another putative HRE is located in intron 1. The presence of a shared HIF binding site (RCGTG) 10 kb upstream and downstream of the ATG revealed a peak 1–5 kb downstream of the ATG in screening Ahsg genes from 15 species, with an average of 2 HREs / 1 kb windows. Notably, not only were annotated human ChIP-seq HIF sites located within this peak, but four potential mouse HREs were also found. Alignment of the latter with enhancer markers revealed a close association with hypersensitivity to H3K27Ac, ​​H3K4Me1, and DNase I.

[0114] In vitro hypoxia activated fetoglobulin A transcription. Five putative HRE sites from mouse Ahsg, along with their surrounding DNA and nonsense mutations, were cloned into a luciferase reporter plasmid. Figure 4A Normal rat kidney epithelial (NRK) cells transfected with a reporter plasmid containing only the putative -2kbHRE did not show increased luciferase activity under hypoxic conditions. Figure 4B Conversely, NRK cells carrying reporter plasmids containing downstream HREs exhibited significantly increased luminescence under hypoxia. No increased luciferase activity was detected when these HREs were mutated. Furthermore, no enhanced luciferase activity was observed when the upstream and downstream HREs were combined. In summary, the downstream HRE confers hypoxia-inducible luciferase activity to the mouse Ahsg gene. Finally, fetoglobulin A protein was detected in primary mouse PT cells (pPTC), NRK cells, and human HK2 cells when cultured under hypoxic conditions. Figure 4C -E). In summary, these findings identify fetoglobulin A as a novel, evolutionarily conserved HIF-dependent target gene.

[0115] Fetoprotein A deficiency exacerbates CKD progression in hypoxic IUGR kidneys. To investigate how fetoprotein A induction in hypoxic IUGR kidneys has long-term effects on renal function, urinary protein levels and glomerular filtration rate (GFR) were measured in adult mice. Figure 5A (B) Compared to normoxic conditions, 9-week-old mice exposed to fetal hypoxia exhibited decreased GFR and increased proteinuria. Furthermore, assessment of fibrotic tissue remodeling revealed greater collagenous structure (extending deeper into the subcortical region) and the highest levels of fibrotic marker expression in the kidneys of nine fetal hypoxic Ahsg KO mice, compared to hypoxia wt and normoxic controls, respectively (Fig. 5C-G). Importantly, these results clearly demonstrate a more systemic and significant impact on renal function in Ahsg KO mice, exhibiting an additive effect of hypoxia and fetoglobulin A deficiency.

[0116] Fetoglobulin A deficiency promotes the accumulation of calcium mineral particles and macrophages in hypoxic IUGR kidneys. Adult fetoglobulin A KO mice are prone to soft tissue calcification, but no obvious calcification was detected in the kidneys of hypoxic Ahsg KO mice, as assessed by von-Kossa staining. To further test whether fetoglobulin A expression in fetal hypoxic kidneys affects mineralized matrix treatment, the presence of calcium-containing nanoparticles was detected by incubating freshly cut kidney sections from E18.5 embryos with ATTO 488 fluorescently labeled fetoglobulin A (488-FA). Due to the high affinity of fetoglobulin A for calcium phosphate, 488-FA staining is more sensitive than conventional mineral staining protocols for detecting calcium-containing matrix and cellular remnants. Therefore, in the absence of von-Kossa or Alizarin Red staining, positive 488-FA staining also highlights calcium-rich structures, including amorphous calcium phosphate aggregates that often precede obvious calcification. 488-FA staining revealed strong marking of PT in normoxic wt kidneys, a major site of calcium reabsorption and therefore also a site of mineralized matrix processing (Fig. 6A). PT staining intensity decreased in hypoxic wt and increased in hypoxic Ahsg KO, suggesting increased and decreased mineralized matrix turnover, respectively (Fig. 6B, C). Only hypoxic Ahsg KO kidneys also showed a granular staining pattern in the papillary region and, less commonly, in the nephrogenic region of the pericortex (arrows in Fig. 6D-I), indicating substantial mineral deposition in the absence of endogenous fetoglobulin A. Excessive mineral deposits or cell debris frequently appeared at sites of enhanced cell death. Indeed, TUNEL staining confirmed apoptosis in hypoxic Ahsg KO kidneys, but not in normoxic or hypoxic wt kidneys. Immunostaining with cleaved caspase-3 in hypoxic Ahsg KO kidneys further confirmed cell death in these kidneys. Finally, the accumulation of mineralized material triggers an inflammatory response, leading to macrophage infiltration, tissue damage, and fibrosis. Staining for the macrophage marker F4 / 80 revealed significant accumulation of these cells only in hypoxic Ahsg KO kidneys (relative to wt kidneys), suggesting a protective effect of fetoglobulin A (Fig. 6J-N). Overall, these data provide the first illustrative examples of the role of fetoglobulin A in binding to and clearing mineralized matrix in the kidneys and protecting tissue integrity.

[0117] Fetoglobulin A attenuates the expression of hypoxia-induced fibrosis markers by antagonizing TGF-β signaling. Kidneys from fetal hypoxia Ahsg KO mice typically exhibit higher levels of fibrosis marker expression compared to fetal hypoxia wt animals (Figure 5), despite similar mRNA levels of transforming growth factor β-1 (Tgfb1), a potent inducer of fibrosis. These findings were elucidated in vitro using freshly isolated pPTCs. Fibrosis marker expression was generally enhanced in Ahsg KO pPTCs compared to wt cells, and even further increased under hypoxic culture conditions. Figure 7A -D, G). This hypoxia-induced increase was attenuated by supplementing the culture medium with fetoglobulin A (D, G). Figure 7B -G). Stimulation of pPTCs with recombinant TGF-β1 resulted in robust phosphorylation of its intracellular signal transducer Smad3, which was more than three times stronger in Ahsg KO pPTCs than in wt cells. Figure 7H This indicates that Ahsg KO cells respond more actively to TGF-β1. Conversely, the addition of fetoglobulin A prior to TGF-β1 treatment reduced Smad3 phosphorylation. These findings are consistent with previous reports, which described fetoglobulin A as a TGF-β type II receptor mimic and cytokine antagonist. Overall, the results suggest that, in addition to its protective role as a mineral molecular chaperone, fetoglobulin A reduces hypoxia-induced renal fibrosis by directly antagonizing TGF-β1 signaling.

[0118] Fetoglobulin A significantly reduces the expression of fibrosis markers in the kidneys following ischemia-reperfusion injury in vivo. Next, we explored the therapeutic potential of fetoglobulin A supplementation for treating kidney injury experimentally. For this purpose, a well-established mouse model of unilateral ischemia-reperfusion injury was used. As its name suggests, this IRI model optimally mimics ischemia-reperfusion injury in the kidneys following cardiovascular surgery, kidney transplantation, or renal tumor removal. It is direct and reproducible. Ischemia was induced in the left kidney by clamping the renal vessels for 30 minutes, with the right kidney serving as a control. First, the accumulation of calcium mineral particles and macrophages in the hypoxic IUGR kidney showed that ischemia-reperfusion indeed induced the deposition of calcium mineral nanoparticles in the operated kidney, while none were observed in the contralateral control kidney. Figure 8 Furthermore, treatment of IRI mice with fetoglobulin A, compared to 0.9% NaCl, showed that on day 5, fetoglobulin A treatment significantly reduced specific fibrosis markers (collagen Col1a1, Col3a1, and Col6a1) in ischemic damaged kidneys compared to animals treated with 0.9% NaCl. Figure 9A(B, C). Conversely, fetoglobulin A supplementation had no effect on the expression of non-collagenous fibrosis markers Acta2, Fn1, or Vim. Figure 9D (E, F). Therefore, fetoglobulin A supplementation attenuated the expression of fibrosis markers following ischemia-reperfusion injury. In summary, these in vivo experiments clearly establish the role of fetoglobulin A as a mineral molecular chaperone in protecting the kidneys from hypoxia (or IR) injury.

[0119] In mice undergoing ischemia-reperfusion injury (IRI), prophylactic administration of human fetoglobulin A resulted in less tissue damage and reduced fibrotic remodeling. 900 μg of fetoglobulin A was administered intravenously 24 hours and (again) 3 hours before unilateral IRI (20 minutes of ischemia) in the left kidney. PBS was administered to control animals. Kidneys were harvested 24 hours post-surgery, with the right kidney used as a control. In IRI kidneys pretreated with fetoglobulin A, the early damage marker Kim1 (… Figure 10A Krt18 Figure 10B ), Krt20 ( Figure 10C ) and Lcn2( Figure 10D The expression level of fetoglobulin A was reduced. This has shown that pretreatment with fetoglobulin A alleviates tissue damage in reperfusion injury (IRI). Additionally, a decrease in Vim expression was observed. Figure 10G This indicates less epithelial-mesenchymal transition (EMT). Collagen remodeling is a relatively late event during fibrotic remodeling, and it does not play a significant role within 24 hours after IRI, thus Col1a1 ( Figure 10H ), Col3a1 ( Figure 10I ) and Col6a1( Figure 10J No unexpected changes were observed. In mice pretreated with fetoglobulin A, Arg1 ( Figure 10K Arg1 expression was suppressed, indicating a reduced demand for collagen synthesis. Arg1 is important for increased collagen synthesis because it is required to supply L-proline (a major component of collagen). Furthermore, pretreatment with fetoglobulin A reduced the activity of the chemokine Ccl2 (Ccl2), which is involved in damage signaling and repair. Figure 10L ), Il-6 ( Figure 10M ) and Tnf-α ( Figure 10N The expression of ). In summary, these experiments (especially early damage markers) show that prophylactic administration of human fetoglobulin A in mice experiencing ischemia-reperfusion injury (IRI) results in less damage and reduced fibrotic remodeling, even at relatively low fetoglobulin A concentrations.

[0120] Exogenous fetoglobulin A was present in the kidney tissue of mice after intravenous (iv) administration. Human fetoglobulin A was not detected in samples injected with PBS. Figure 11A The presence of sc-133146-HRP (in lane GL) confirms the specificity of this anti-fetoprotein A antibody against human fetoprotein A, but not mouse fetoprotein A. IRI kidneys injected with PBS ( Figure 11A The JL lane showed the highest abundance of mouse fetoglobulin A in all samples, indicating high biomineralization and tissue damage in these kidneys. Kidney samples from mice injected with human fetoglobulin A protein showed strong bands for human fetoglobulin A. Figure 11A The presence of endogenous mouse fetoglobulin A in kidney tissue following intravenous administration (AF) confirmed its presence. Compared to all other samples, endogenous mouse fetoglobulin A was elevated only in the IRI kidneys injected with PBS, indicating significant biomineralization and tissue damage in these kidneys. Figure 11B Human fetal protein A was not detected in kidney samples injected with PBS, confirming the specificity of the DFTA00 ELISA kit from R&D Systems (bio-techne) for human fetal protein A but not mouse fetal protein A. Figure 11C IRI kidneys injected with human fetoglobulin A (HFA) showed higher tissue levels of HFA, indicating a greater degree of biomineralization in ischemic kidneys compared to untreated control kidneys. Figure 11C Following intravenous administration, exogenous fetoglobulin A was observed in the proximal tubules of mice. Figure 11D -F). Without being bound by any theory, it can be hypothesized that fetoglobulin A may be drawn from the blood to the site of tissue damage, thereby combating further tissue destruction and reducing its concentration in the blood. In summary, these experiments show that intravenous administration of fetoglobulin A leads to an increase in fetoglobulin A in kidney tissue.

[0121] The time course of human fetoglobulin A in mouse serum after intravenous administration. One day later, the amount of human fetoglobulin A detected in serum was reduced compared to 1 minute or 1 hour after injection. Figure 12A Furthermore, human fetal protein A was undetectable after one week. The lack of detection of mouse fetal protein A with the anti-human fetal protein A antibody sc-133146-HRP can be inferred from the fact that animals injected with PBS did not give a signal. Standardized ELISA data using the human fetal protein A-specific DFTA00 ELISA kit showed a decrease in intravenously injected human fetal protein A in mouse serum samples. Figure 12B(C). Compared to the injection time point (1 minute), the amount of circulating human fetoglobulin A decreased to 80% after 1 hour and to 8.5% after 1 day. After 1 week, human fetoglobulin A was no longer detectable, similar to the control mice injected with PBS (black circles). In conclusion, these experiments show that daily injections are necessary to maintain the presence of circulating human fetoglobulin A.

[0122] method

[0123] Animals. Breeding, genotyping, and all animal experiments were conducted in accordance with Swiss law on animal welfare and with the approval of the local authorities (Bern cantons BE96 / 11, BE105 / 14, and BE105 / 17). All mice, including Ahsg... tm1Mbl Mice and Clcn5 tm1Gug Mice were maintained in a C57BL / 6 background and housed in IVC cages with free access to food and water, and underwent a 12-hour day / night cycle.

[0124] Induction of hypoxia in pregnant mice. For pre-mating, female mice in breeding were checked daily morning by vaginal plugging, and if present, this time point was set as day of gestation (E) 0.5. Ahsg KO mice were obtained from heterozygous breeding pairs, which also produced heterozygous and wt littermates, which served as controls. Daily food consumption (weight difference between the initial food provided and the food remaining after 24 hours) and maternal weight gain from E0.5 to E18.5 were recorded during pregnancy. For induction of hypoxia, pregnant mice at E13.5 were transferred to a hypoxia glove sterile operating cabinet (Coy Laboratory Products, Grass Lake, USA). The following day, oxygen levels were gradually reduced to 10% over 6–8 hours, with intermittent pauses at 16% and 12.5% ​​to acclimate the animals to the increasing hypoxic conditions. Adequate air circulation was maintained in the room with fans. Low CO2 levels were maintained by chelating excess CO2 in a box filled with soda lime (Sigma, 72073) connected to the air circulation system. Excess moisture was absorbed using silica gel orange granules (Sigma, 1.01969) (changed daily). Mice in the caloric control group received a diet matched to the average food consumption of mice in the hypoxia group. Pregnant hypoxic or control mice were euthanized at E18.5, and fetuses and placentas were collected, weighed, and prepared for further analysis. Fetal kidneys were dissected in PBS using a Leica M80 stereomicroscope. Kidneys from 3–4 week old normoxic mice were isolated for primary proximal tubular cell (pPTC) preparation. Renal function (GFR and proteinuria) was assessed in 9-week-old hypoxic or normoxic fetuses.

[0125] Ischemia induction and administration for therapeutic treatment experiments. For use in... Figure 8 In the experiment described in Figure 9, mice were anesthetized with isoflurane. Midline laparotomy was performed in the deeply anesthetized animals. Ischemia was induced in the left kidney by clamping the renal vessels for 30 minutes, with the right kidney serving as a control (Figure 9). Immediately after surgery, mice received their first treatment via intraperitoneal injection of either physiological NaCl solution or bovine fetoglobulin A monomer (100 μg / g body weight). Injections were repeated daily for 4 days. Kidneys were isolated and analyzed on day 5. NaCl solution: Natrium Chloratum (Bichsel) 0.9% (154 mM) (REF 100 0 090, Bichsel, Interlaken, CH). Fetoglobulin A solution: 0.338 EU / ml LPS, 10 mM Na3PO4, 8 g / L NaCl (137 mM).

[0126] Ischemia induction and administration for prophylactic treatment (prevention) experiments. For the experiments in Figure 10, mice were intravenously injected with 900 μg of fetoglobulin A 24 hours and 3 hours before unilateral ischemia. Control animals were injected intravenously with PBS. For ischemia induction, mice were anesthetized with isoflurane. In deeply anesthetized animals, midline laparotomy was performed, and ischemia was induced in the left kidney by clamping the renal vessels for 20 minutes. The right kidney served as a control without surgery. Kidneys were collected 24 hours post-surgery. The average weight of the mice used in the experiments in Figure 10 was 22 g. PBS solution: 2.67 mM KCl, 1.47 mM KH2PO4, 137.93 mM NaCl, 8.06 mM Na2HPO4-7xH2O.

[0127] Transcutaneous assessment of glomerular filtration. Glomerular filtration rate (GFR) was determined in conscious animals as described in Schreiber, A. et al., Transcutaneous measurement of renal function in conscious mice. Am J Physiol Renal Physiol 303, F783-788 (2012). Briefly, plasma clearance of FITC-sinistrin (Fresenius-Kabi, LI9830076) was measured across the skin using a light-emitting diode with maximum emission of FITC at 470 nm and a photodiode with maximum sensitivity for detecting fluorescence at 525 nm. The decrease in fluorescence intensity over time was then converted to GFR.

[0128] Proteinuria. Urinary protein content was determined using the Bradford assay. 3 μl of urine was mixed with 150 μl of 1x Bradford reagent, incubated at room temperature for 5 minutes, and the absorbance was measured at 595 nm. (5x Bradford reagent was prepared by dissolving 50 mg of Brilliant Blue G-250 (Sigma, B-1131) in 24 mL of ethanol and 50 mL of 85% phosphoric acid, then adjusting the total volume to 100 mL with ultrapure water). Urinary creatinine content was determined using the Jaffe method. 10 μl of a 1:10 diluted urine solution was mixed with 100 μl of creatinine reagent, incubated at room temperature for 10 minutes, and the absorbance was measured at 510 nm. (Cretinine reagent composition: 10 mM picric acid and 250 mM NaOH, pH 13). Finally, the protein / creatinine ratio was calculated for each sample.

[0129] Glomerular Counting. 100μm Z-stacked images of whole E18.5 kidneys stained with nephrin (R&D AF3159) were analyzed using the open-source image processing software Fiji (ImageJ, version 2.0.0-rc69 / 1.52i, https: / / imagej.net / Fiji). In the TrackMate v3.8.0 plugin, the downsampled LoG detector was set to 80.0 pixels, the threshold to 16 pixels, and the downsampling factor to 2 for the estimated speckle diameter. The number of points per frame was added to the mice to count the number of glomeruli per kidney. Considering the average glomerular diameter of 80μm, a 100μm distance between frames was chosen to avoid repeatedly counting the same glomeruli in consecutive images.

[0130] Microarray analysis. Total RNA was isolated from the kidneys of male E18.5 controls under hypoxia, normoxic, and caloric conditions using the RNeasy Mini kit (Qiagen, 74104). Only high-quality RNA (RIN>8, 260 / 280 ratio>2, 260 / 230 ratio>1.8) was used for further analysis. The WT Expression Kit (4411973, Life Technologies) was used to process 100 ng of total RNA sample. 5.5 μg of cDNA was fragmented and... Label using the WT TerminalLabeling kit (901525, Affymetrix). Follow the instructions on the Affymetrix website. The experimental protocol described in the expression analysis manual (Fluidics protocol FS450_0007) was followed, in which 2.3 μg of biotinylated fragment was hybridized with an Affymetrix Mouse Gene 1.0ST array at 45°C for 16 hours, washed, and stained. Affymetrix was then used... The array was scanned using a Scanner 3000 7G, and raw data were extracted from the scanned images and analyzed using the Affymetrix Power Tools software package. Hybridization quality was assessed using Affymetrix Expression Console software (version 1.1.2800.28061). Normalized expression signals were calculated from Affymetrix CEL files using the Robust Multi-array Average (RMA) algorithm. Differential hybridization characteristics were identified using the R Bioconductor package “limma” (which performs linear models for microarray data). P-values ​​were adjusted for multiple tests using the Benjamini and Hochberg methods to control for false discovery rate (FDR). Probe groups showing at least a 1.3-fold change and FDR < 0.05 were considered significant. Differential expression values ​​were plotted in the kidneys of E18.5 controls under hypoxia, normoxic, and caloric conditions using Heatmapper (http: / / www.heatmapper.ca) with mean linkage and Euclidean distance measurements. For feature annotations, GO-term analysis was performed using the DAVID platform (https: / / david.ncifcrf.gov).

[0131] RT-qPCR. Follow the manufacturer's experimental protocol. Total RNA was isolated using reagents (Invitrogen 15596026). RNA concentration and quality were determined using a Nanodrop 1000 spectrophotometer (ThermoFisher Scientific, Switzerland), and 1000 ng was transcribed into cDNA using a PrimeScript RT Reagent Kit (Takara, RR037A). The cDNA was diluted to 2 ng / μl, and qPCR was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems) using TaqMan Gene Expression Assays (ThermoFisher) or FAM-labeled UPL probes (Roche) with appropriate gene-specific primers and a TaqMan Fast Universal PCR Master mix (Applied Biosystems, 4352042). Data analysis was performed using Microsoft Excel. (-ΔCt) The method is used to calculate relative expression levels for RT-qPCR.

[0132] Identification of HIF binding sites. To identify putative HIF binding sites, the JASPAR database (http: / / jaspar.genereg.net) was used to analyze 20kb genomic regions (10kb upstream and downstream of the start codon) overlapping with the Ahsg locus in 15 different species (cats, chickens, chimpanzees, cattle, dogs, ghost sharks, horses, humans, mice, pigs, rabbits, rats, sheep, African clawed frogs, and zebrafish). The relative profile score threshold was set to 90%. For enrichment analysis, putative sites from all species were clustered in a 1kb window. HREs with relative scores >0.9, >0.93, and >0.97 are shown. For human HIFα sites identified by ChIP-seq in hypoxic MCF7 cells (see [link to relevant documentation]). J, Oikonomopoulos S, Ragoussis J, Pugh CW et al., Blood 2011 Jun 9, 117(23):e207-17.; Series GSE28352; https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE28352) was compared with the activity regulatory markers of the AHSG locus. The HIF-1-α and HIF-2-α datasets covering chr3:180,000,000-190,000,000 (including the AHSG locus) were converted into BAM files using the web-based Galaxy platform (https: / / usegalaxy.org) and uploaded to the human assembly GRCh37 / hg19 on the USCS Genome Browser (https: / / genome.ucsc.edu). For this comparison, the following dataset was added: layered chromatin markers frequently found near activity regulatory elements (H3K27Ac and H3K4Me1, ENCODE) in seven cell lines and near open chromatin (DNase IHS, ENCODE) in hypoxic MCF7 cells. For the mouse Ahsg locus, potential HIF binding sites identified using JASPAR were compared with datasets derived from the liver, heart, and kidney of 8-week-old mice (DNase IHS, ENCODE / UW; H3K27Ac and H3K4Me1, ENCODE / LICR), where mean signal intensity is shown (bar graphs, auto-scaled, log-transformed, smoothed (16 pixels)).

[0133] Molecular cloning. For the luciferase assay, specific primers and... GXL DNA polymerase (Takara, R050A) was used to amplify a 2.5 kb promoter fragment upstream of mouse Ahsg ATG from genomic DNA (C57BL / 6). A 500 bp promoter fragment (wt and mutant) and a 500 bp intron sequence fragment (wt and mutant) were synthesized using IDTDNA (https: / / eu.idtdna.com / pages). The promoter fragment was inserted into the pGL3-basic-P2P-607 plasmid using NcoI and SacI restriction enzymes (both from NEB, R3193S and R3156S). The intron fragment was inserted into the pGL3-basic vector or a pGL3-basic vector carrying the promoter fragment using BamHI and SalI restriction enzymes (both from NEB, R3136S and R3138S). For in situ hybridization, cDNA from exons 2–5 of mouse Ahsg was obtained from IDTDNA and cloned into pBluescriptII KS- using SpeI and EcoRI restriction enzymes (both derived from NEB, R3133S and R3101S).

[0134] Luciferase assay. 24 hours post-transfection, cells were washed twice with PBS, lysed on ice (250 mM KCl, 50 mM Tris / H3PO4 pH 7.8, 10% glycerol, 0.1% NP40) for 20 min, and centrifuged at 14,000 rpm for 10 min at 4°C. 10 μl of supernatant was used for each reaction. Injection and activity measurements of the reaction solutions (luciferase: 100 μl of 25 mM Tris / H3PO4 pH 7.8, 10 mM MgSO4, 2 mM ATP pH 7.5, 50 μM luciferin; renilla: 100 μl of 50 mM Tris / HCl pH 7.6, 100 mM NaCl, 1 mM EDTA, 0.5 μM coelenterazine) were performed using a Fluoroskan Ascent FL (ThermoFisher). Each sample was measured in duplicate, and luciferase activity was normalized by renal gill activity.

[0135] In situ hybridization was performed using whole-unit anaerobic digestion. E18.5 kidneys were fixed in 4% PFA, dehydrated, and stored in methanol at -20°C. In situ hybridization was performed using digitoxin-labeled RNA probes as described in Rudloff, S. and Kemler, R. Differential requirements for beta-catenin during mouse development. Development 139, 3711-3721 (2012). Probes were generated using a DIG RNA-labeled mixture (Roche, 11175025910) and T3 or T7 RNA polymerases (both from Roche, 11031163001 or 10881767001). Antibodies conjugated with alkaline phosphatase were used to detect the DIG-labeled probes (Roche, 11093274910).

[0136] TUNEL staining. The Promega DeadEnd Colorimetric TUNEL System (G7360) was used to detect fragmented DNA in apoptotic cells, as per the manufacturer’s instructions.

[0137] Histochemistry. For immunohistochemistry, PFA-fixed, paraffin-embedded tissue sections were rehydrated and endogenous peroxidase was blocked by incubating slides in the dark at room temperature in 1.5% H2O2 solution (0.02M citric acid, 0.06M Na2HPO4) for 15 minutes. Antigen extraction was performed by boiling in Tris-EDTA buffer pH 9 for 20 minutes, followed by slow cooling to room temperature. After blocking in 2% BSA in PBS for 1 hour at room temperature, sections were incubated overnight with primary antibody in blocking solution at 4°C. After three washes in PBS, sections were incubated with HRP-conjugated secondary antibody (mouse or rabbit: Dako EnVision+System (K4001 or K4003) from Agilent; goat: SCBT, sc-2304) at room temperature for 1 hour. After three washes in PBS, signals were visualized using DAB (Agilent, K3468). Counterstained sections with Harris hematoxylin solution (Sigma, HHS16), dehydrated, and mounted using Eukitt medium (Sigma, 03989). For Sirius red staining of collagen, dewaxed, rehydrated tissue sections were incubated at room temperature for 1 hour in staining solution (0.5 g Direct Red 80 in saturated aqueous picric acid solution (both from Sigma, 365548 and P6744)). After washing twice in acidified water (0.5% glacial acetic acid), sections were dehydrated and mounted using Eukitt.

[0138] Immunofluorescence staining. Frozen sections were fixed in 4% PFA for 10 minutes at room temperature, washed twice in PBS, and permeabilized by incubation in PBST (0.1% Triton X-100 in PBS) for 10 minutes at room temperature. After blocking for 1 hour in 10% FCS and 0.5% Tween-20 in PBS at room temperature, sections were incubated overnight at 4°C with primary antibody in blocking solution. After three washing steps in PBS, sections were incubated in the dark at room temperature for 1 hour with fluorescently conjugated secondary antibody in blocking solution. DNA was stained with DAPI 1:5000 in PBS. Sections were mounted in MOWIOL solution (2.4g MOWIOL 4-88 reagent (Merck, 475904) in 6g glycerol and 18ml 0.13M Tris pH 8.5). For whole-frame immunofluorescence staining of E18.5 kidneys, the iDisco staining protocol with methanol pretreatment (https: / / idisco.info / idisco-protocol / ) was used. Incubation time n = 1 day and solution volume 1.6 ml were used for the relevant steps. The kidneys were placed in 8-well glass slides (ThermoFisher, 154534) and imaged immediately.

[0139] Fluorescence detection of calcium. Thick frozen sections (30 μm) were incubated in the dark at room temperature for 60 min with 10 ng / ml ATTO 488 fluorescently labeled fetoglobulin A (in calcium-free PBS), washed three times with PBS, and mounted with MOWIOL solution. Cell nuclei were counterstained with DAPI.

[0140] Imaging. Polychrome V light source, Orca-R from Hamamatsu. 2 Fluorescence imaging was performed on an IMIC digital microscope (FEI, model 4001) using a camera controller (C10600) and real-time acquisition software (FEI, version 2.6.0.14). Image analysis was performed using offline analysis software (FEI). Bright-field imaging was performed on a Nikon E600 microscope equipped with Nikon objectives (Plan Fluor ELWD 20x / 0.45, Plan Apo 40x / 1.0Oil, and 60x / 1.40Oil) using a Digital Sight DS-UE camera controller and a DSRi1 camera (both from Nikon). Image analysis was performed using Nikon software NISElements 4.0.

[0141] ELISA. Mouse fetoglobulin A levels (e.g., in serum or kidney tissue) were measured using the Mouse-Specific Fetoglobulin A / AHSG Quantikine ELISA Kit (R&D, MFTA00) as directed by the manufacturer, Bio-Techne. Human fetoglobulin A levels (e.g., in serum or kidney tissue) were measured using the Human-Specific Fetoglobulin A / AHSG Quantikine ELISA Kit (R&D, DFTA00) as directed by the manufacturer, Bio-Techne.

[0142] Cell culture. Normal rat kidney (NRK) cell lines were cultured in DMEM (Gibco, 41965-039) and 10% fetal bovine serum (FBS). Human kidney (HK-2) cell lines were cultured in keratinocyte-SFM medium (Gibco, 17005-075). For the luciferase assay, [the following was performed using...]. NRK cells were transfected with luciferase reporter plasmid and pCMV-nephrotic gill (10% of total transfected DNA, used for normalization) using reagent (Polyplus, 114-07). Cells were stimulated with 1 mM DMOG (Echelon Biosciences, F-0010) 6 hours post-transfection and harvested 24 hours post-transfection. Cell cultures were incubated at 0.2% oxygen for 48 hours for hypoxia. Primary proximal tubular cells (pPTCs) were isolated from 3–4 week old kidneys (see Terryn, S. et al., A primary culture of mouse proximal tubular cells, established on collagen-coated membranes. Am J Physiol Renal Physiol 293, F476-485 (2007)). Briefly, pPTC fragments were obtained by digesting cortical kidney tissue with collagenase and filtering through an 80 μm pore membrane. pPTCs were cultured in DMEM / F12 (Gibco, 21041-025) supplemented with 15 mM HEPES, 0.55 mM sodium pyruvate, 1% NEAA, and renal epithelial cell growth medium (REGM) supplement (Lonza, CC-4127). Serum from Ahsg KO mice was used instead of FBS. After confluence, pPTCs were separated and replated once using Accutase solution (Sigma, A6964). Cell culture was performed at 0.2% oxygen for 48 hours under 24-hour hypoxia. 100 μg / ml fetoglobulin A (Sigma, F3385) or bovine serum albumin (BSA, Sigma, A3059) was added to the medium 48 hours before the end of the experiment. Cells were starved for 24 hours before treatment with 5 ng / ml rmTGF-β1 (R&D, 7666-MB) for 5 minutes.

[0143] Western blot analysis. Total protein lysates were obtained using RIPA buffer (Sigma, R0278) supplemented with a protease inhibitor (Roche, 11836153001). Proteins were separated by SDS-PAGE and blotted onto PVDF membranes (ThermoFisher, 88518). After blocking with 5% emulsion in TBST, the membranes were incubated overnight at 4°C with primary antibody, followed by incubation at room temperature for 1 hour with HRP-conjugated secondary antibody. Signal intensity was detected using ECL (GE Healthcare, RPN2106) or SuperSignal (ThermoFisher, 34076). Densitometry analysis was performed using the open-source image processing software Fiji (ImageJ, version 2.0.0-rc69 / 1.52i, https: / / imagej.net / Fiji).

[0144] Calculation of the concentration of bovine fetoglobulin A injected intraperitoneally in mice. The reference blood volume for mice is 58.5 ml / kg (nc3rs.org.uk) and 77-80 ml / kg (jax.org). Based on these values, 68.5 ml / kg was used for calculations. The calculated molecular weight of bovine fetoglobulin A (UniProtKB-P12763(FETUA_BOVIN)) is 38.4 kDa. Therefore, if all injected fetoglobulin A is in the bloodstream or if intravenous injection of fetoglobulin A is performed, a blood concentration of 38 μM will be achieved. Furthermore, fetoglobulin A undergoes post-translational modifications. Here, glycosylation plays a dominant role, increasing the molecular weight to 51-67 kDa. Therefore, a molecular weight of 60 kDa (where 1 Da = 1 g / mol) was used in the calculations. This resulted in a concentration of 24.3 μM in the blood of injected mice, if all injected fetoglobulin A were in the blood or if intravenous injection of fetoglobulin A was performed, based on an injection dose of 100 μg / g body weight:

[0145]

[0146] However, intraperitoneal injection results in lower blood concentrations because fetoglobulin A must be transported from the peritoneal cavity into the circulation. During this process, not all molecules are absorbed, due to partial degradation in the lumen or pinocytosis and metabolism by mesothelial cells. In one study (Regoeczi et al. (1989), Am JPhysiol., 256(4Pt1):E447-52), it was shown that after 2–3 hours, intraperitoneally injected albumin reached approximately 40% of the stated dose in the blood compared to intravenous injection of the same amount. Based on 40% uptake in mice, the concentration obtained after intraperitoneal injection was:

[0147]

[0148] Therefore, if 100 mg / kg body weight of fetoglobulin A is injected intraperitoneally, the resulting blood concentration will be approximately 9.7 μM. As described above, such relatively low doses have been found sufficient to treat ischemic kidney damage.

[0149] Calculations of the concentration of intravenously injected human fetoglobulin A in mice. The reference blood volume for mice was 58.5 ml / kg (nc3rs.org.uk) and 77-80 ml / kg (jax.org). Based on these values, 68.5 ml / kg was used for calculations. The calculated molecular weight of human fetoglobulin A (UniProtKB-P12763(FETUA_BOVIN)) was 38.4 kDa. Furthermore, fetoglobulin A undergoes post-translational modifications. Here, glycosylation plays a dominant role, increasing the molecular weight to 51-67 kDa. Therefore, a molecular weight of 60 kDa was used in the calculations. In experiments used for prophylactic treatment, the average weight of mice was 22 g. For mice weighing 22 g, the corresponding blood volume was approximately...

[0150]

[0151] When 900 μg of human placental protein A is injected, this results in a blood concentration of [missing value].

[0152]

[0153] Therefore, if 900 μg of human fetal globulin A is injected intravenously into mice with an average weight of 22 g, the resulting blood concentration will be approximately 9.9 μM. As described above, such a relatively low dose has been found to be sufficient to prevent ischemic kidney damage.

[0154] Concentrations and dosage calculations of human fetoglobulin A in humans. The reference blood volume for adults is 77 ml / kg (male) and 65 ml / kg (female), with an average volume of 71 ml / kg. The molecular weight of human fetoglobulin A (UniProtKB-P02765(FETUA_HUMAN)) is generally comparable to that of bovine fetoglobulin A, approximately 60 kDa (glycosylated). The blood concentrations obtained after administering 10 mg / kg (iv), 25 mg / kg (iv), and 50 mg / kg (iv) of fetoglobulin A to adults would be:

[0155]

[0156]

[0157]

[0158] Similarly, it can be calculated which dose corresponds to the 9.7 μM concentration mentioned above:

[0159]

[0160] Similarly, it can be calculated which dose corresponds to the 9.9 μM concentration mentioned above:

[0161]

[0162] Therefore, the calculated equivalent amount of fetoglobulin A administered intraperitoneally to mice at a dose of 100 μg / g body weight is approximately 41.3 mg / kg when administered intravenously to adults; and the calculated equivalent amount of fetoglobulin A administered intravenously to mice weighing 22g is approximately 42.2 mg / kg when administered intravenously to adults.

[0163] Data Analysis. Statistical analysis and graphing were performed using Prism 7 (https: / / www.graphpad.com). Two groups were compared using t-tests, and multiple groups were compared using one-way ANOVA (Tukey multiple comparison test, unless otherwise specified). ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant; error bars, standard deviation (SD); whiskers, minimum to maximum. sequence list <110> Universitaet Bern <120> Fetoprotein A used to treat kidney disease <130> 2020_CH001_A317 <150> EP20162490.5 <151> 2020-03-11 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 368 <212> PRT <213> Human <400> 1 Met Lys Ser Leu Val Leu Leu Leu Cys Leu Ala Gln Leu Trp Gly Cys 1 5 10 15 His Ser Ala Pro His Gly Pro Gly Leu Ile Tyr Arg Gln Pro Asn Cys 20 25 30 Asp Asp Pro Glu Thr Glu Glu Ala Ala Leu Val Ala Ile Asp Tyr Ile 35 40 45 Asn Gln Asn Leu Pro Trp Gly Tyr Lys His Thr Leu Asn Gln Ile Asp 50 55 60 Glu Val Lys Val Trp Pro Gln Gln Pro Ser Gly Glu Leu Phe Glu Ile 65 70 75 80 Glu Ile Asp Thr Leu Glu Thr Thr Cys His Val Leu Asp Pro Thr Pro 85 90 95 Val Ala Arg Cys Ser Val Arg Gln Leu Lys Glu His Ala Val Glu Gly 100 105 110 Asp Cys Asp Phe Gln Leu Leu Lys Leu Asp Gly Lys Phe Ser Val Val 115 120 125 Tyr Ala Lys Cys Asp Ser Ser Pro Ala Asp Ser Ala Glu Asp Val Arg 130 135 140 Lys Val Cys Gln Asp Cys Pro Leu Leu Ala Pro Leu Asn Asp Thr Arg 145 150 155 160 Val Val His Ala Ala Lys Ala Ala Leu Ala Ala Phe Asn Ala Gln Asn 165 170 175 Asn Gly Ser Asn Phe Gln Leu Glu Glu Ile Ser Arg Ala Gln Leu Val 180 185 190 Pro Leu Pro Pro Ser Thr Tyr Val Glu Phe Thr Val Ser Gly Thr Asp 195 200 205 Cys Val Ala Lys Glu Ala Thr Glu Ala Ala Lys Cys Asn Leu Leu Ala 210 215 220 Glu Lys Gln Tyr Gly Phe Cys Lys Ala Thr Leu Ser Glu Lys Leu Gly 225 230 235 240 Gly Ala Glu Val Ala Val Thr Cys Met Val Phe Gln Thr Gln Pro Val 245 250 255 Ser Ser Gln Pro Gln Pro Glu Gly Ala Asn Glu Ala Val Pro Thr Pro 260 265 270 Val Val Asp Pro Asp Ala Pro Pro Ser Pro Pro Leu Gly Ala Pro Gly 275 280 285 Leu Pro Pro Ala Gly Ser Pro Pro Asp Ser His Val Leu Leu Ala Ala 290 295 300 Pro Pro Gly His Gln Leu His Arg Ala His Tyr Asp Leu Arg His Thr 305 310 315 320 Phe Met Gly Val Val Ser Leu Gly Ser Pro Ser Gly Glu Val Ser His 325 330 335 Pro Arg Lys Thr Arg Thr Val Val Gln Pro Ser Val Gly Ala Ala Ala 340 345 350 Gly Pro Val Val Pro Pro Cys Pro Gly Arg Ile Arg His Phe Lys Val 355 360 365 <210> 2 <211> 1107 <212> DNA <213> Human <400> 2 atgaagtccc tcgtcctgct cctttgtctt gctcagctct ggggctgcca ctcagcccca 60 catggcccag ggctgattta tagacaaccg aactgcgatg atccagaaac tgaggaagca 120 gctctggtgg ctatagacta catcaatcaa aaccttcctt ggggatacaa acacaccttg 180 aaccagattg atgaagtaaa ggtgtggcct cagcagccct ccggagagct gtttgagatt 240 gaaatagaca ccctggaaac cacctgccat gtgctggacc ccacccctgt ggcaagatgc 300 agcgtgaggc agctgaagga gcatgctgtc gaaggagact gtgatttcca gctgttgaaa 360 ctagatggca agttttccgt ggtatacgca aaatgtgatt ccagtccagc agactcagcc 420 gaggacgtgc gcaaggtgtg ccaagactgc cccctgctgg ccccgctgaa cgacaccagg 480 gtggtgcacg ccgcgaaagc tgccctggcc gccttcaacg ctcagaacaa cggctccaat 540 tttcagctgg aggaaatttc ccgggctcag cttgtgcccc tcccaccttc tacctatgtg 600 gagtttacag tgtctggcac tgactgtgtt gctaaagagg ccacagaggc agccaagtgt 660 aacctgctgg cagaaaagca atatggcttt tgtaaggcaa cactcagtga gaagcttggt 720 ggggcagagg ttgcagtgac ctgcatggtg ttccaaacac agcccgtgag ctcacagccc 780 caaccagaag gtgccaatga agcagtcccc acacccgtgg tggacccaga tgcacctccg 840 tcccctccac ttggcgcacc tggactccct ccagctggct cacccccaga ctcccatgtg 900 ttactggcag ctcctccagg acaccagttg caccgggcgc actacgacct gcgccacacc 960 ttcatgggtg tggtctcatt ggggtcaccc tcaggagaag tgtcgcaccc ccggaaaaca 1020 cgcacagtgg tgcagcctag tgttggtgct gctgctgggc cagtggttcc tccatgtccg 1080 gggaggatca gacacttcaa ggtctag 1107 <210> 3 <211> 359 <212> PRT <213> Bovine <400> 3 Met Lys Ser Phe Val Leu Leu Phe Cys Leu Ala Gln Leu Trp Gly Cys 1 5 10 15 His Ser Ile Pro Leu Asp Pro Val Ala Gly Tyr Lys Glu Pro Ala Cys 20 25 30 Asp Asp Pro Asp Thr Glu Gln Ala Ala Leu Ala Ala Val Asp Tyr Ile 35 40 45 Asn Lys His Leu Pro Arg Gly Tyr Lys His Thr Leu Asn Gln Ile Asp 50 55 60 Ser Val Lys Val Trp Pro Arg Arg Pro Thr Gly Glu Val Tyr Asp Ile 65 70 75 80 Glu Ile Asp Thr Leu Glu Thr Thr Cys His Val Leu Asp Pro Thr Pro 85 90 95 Leu Ala Asn Cys Ser Val Arg Gln Gln Thr Gln His Ala Val Glu Gly 100 105 110 Asp Cys Asp Ile His Val Leu Lys Gln Asp Gly Gln Phe Ser Val Leu 115 120 125 Phe Thr Lys Cys Asp Ser Ser Pro Asp Ser Ala Glu Asp Val Arg Lys 130 135 140 Leu Cys Pro Asp Cys Pro Leu Leu Ala Pro Leu Asn Asp Ser Arg Val 145 150 155 160 Val His Ala Val Glu Val Ala Leu Ala Thr Phe Asn Ala Glu Ser Asn 165 170 175 Gly Ser Tyr Leu Gln Leu Val Glu Ile Ser Arg Ala Gln Phe Val Pro 180 185 190 Leu Pro Val Ser Val Ser Val Glu Phe Ala Val Ala Ala Thr Asp Cys 195 200 205 Ile Ala Lys Glu Val Val Asp Pro Thr Lys Cys Asn Leu Leu Ala Glu 210 215 220 Lys Gln Tyr Gly Phe Cys Lys Gly Ser Val Ile Gln Lys Ala Leu Gly 225 230 235 240 Gly Glu Asp Val Arg Val Thr Cys Thr Leu Phe Gln Thr Gln Pro Val 245 250 255 Ile Pro Gln Pro Gln Pro Asp Gly Ala Glu Ala Glu Ala Pro Ser Ala 260 265 270 Val Pro Asp Ala Ala Gly Pro Thr Pro Ser Ala Ala Gly Pro Pro Val 275 280 285 Ala Ser Val Val Val Gly Pro Ser Val Val Ala Val Pro Leu Pro Leu 290 295 300 His Arg Ala His Tyr Asp Leu Arg His Thr Phe Ser Gly Val Ala Ser 305 310 315 320 Val Glu Ser Ser Ser Gly Glu Ala Phe His Val Gly Lys Thr Pro Ile 325 330 335 Val Gly Gln Pro Ser Ile Pro Gly Gly Pro Val Arg Leu Cys Pro Gly 340 345 350 Arg Ile Arg Tyr Phe Lys Ile 355 <210> 4 <211> 1080 <212> DNA <213> Bovine <400> 4 atgaaatcct tcgttctgct cttttgcctg gctcagctct ggggctgcca ctcgatcccg 60 cttgacccgg ttgcaggtta taaggaaccg gcctgtgatg acccagacac agagcaagca 120 gccttggctg ccgtggacta catcaacaag caccttcctc ggggctacaa gcacaccttg 180 aaccagattg acagtgtgaa ggtgtggccg aggcggccca cgggagaggt gtatgacatt 240 gaaatagata ccctggaaac cacctgccac gtactggacc ccacgcccct ggcgaactgc 300 agcgtgaggc agcagacgca gcacgcggtg gaaggagact gcgatatcca cgtgctgaaa 360 caagatggcc agttttccgt gctgtttaca aaatgtgatt ccagtccaga ttccgccgag 420 gacgtgcgca agttgtgccc agactgcccc ctgctggcgc cactcaacga cagccgggtg 480 gtgcacgcag tggaggtcgc gctggctacc ttcaatgccg agagcaacgg ctcctactta 540 cagctggtgg aaatttctcg ggctcaattt gtgcctcttc cagtttctgt ctctgtggag 600 tttgcagtgg ctgctactga ctgtattgct aaagaagtcg tagatccaac caagtgcaac 660 ctactggcag aaaagcaata tggcttctgt aaggggtcag tcattcagaa agctcttggt 720 ggggaggacg tcagagtgac ttgcacgttg ttccaaacgc agcctgtgat tccgcagccc 780 cagcccgacg gcgccgaggc tgaggcccca agcgctgtgc cggacgcagc tgggcctacg 840 ccttctgcag ctggcccgcc cgtggcctcc gtggtggtgg ggccaagcgt ggtagcagtt 900 cccctgccgc tgcaccgagc acactacgac ttgcgccaca ctttctccgg ggtggcctca 960 gtggagtcat cctcgggaga agcgttccac gtgggcaaaa cacccatagt ggggcagcct 1020 agcattcctg gaggtccagt ccgcctttgc ccagggagaa tcagatactt caagatctag 1080

Claims

1. Use of fetoglobulin A (AHSG) in the preparation of a medicament for treating renal disease in a subject in need of such treatment, wherein the renal disease is graft function delay caused by ischemia during kidney transplantation, and wherein the fetoglobulin A comprises SEQ ID NO:

1.

2. The use according to claim 1, wherein the fetoglobulin A is derived from human plasma.

3. The use according to claim 1, wherein the fetoglobulin A is recombinantly expressed.

4. The use according to claim 3, wherein the fetoglobulin A is recombinantly expressed in a host cell system providing post-translational modifications.

5. The use according to claim 4, wherein the post-translational modification is glycosylation.

6. The use according to claim 1, wherein the treatment comprises regulating the level of calcification in kidney tissue.

7. The use according to claim 6, wherein the calcification level is a calcification level associated with hypoxia.

8. The use according to claim 1, wherein the treatment comprises removing calcium mineral deposits from kidney tissue.

9. The use according to claim 8, wherein the treatment comprises removing calcium mineral deposits from the transplanted kidney tissue.

10. Use in the preparation of a pharmaceutical composition comprising fetoglobulin A and optionally at least one pharmaceutically acceptable carrier for the treatment of kidney disease, wherein the kidney disease is graft function delay and the graft function delay is caused by ischemia during kidney transplantation.

11. The use according to claim 10, wherein the fetoglobulin A is defined in any one of claims 1 to 5.

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