Fetuin A for the treatment of renal impairment

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to fetuin A (AHSG) for use in methods for treating renal impairment, wherein an effective amount of fetuin A for treating renal impairment is administered to a subject in need, and in particular the renal impairment is ischemic impairment. The present invention also relates to a pharmaceutical composition for use in the treatment of renal impairment, comprising fetuin A and optionally at least one pharmaceutically acceptable carrier. [Background technology]

[0002] The goal of modern medicine is to provide novel treatments, including new targets, to achieve favorable outcomes for specific patient groups. These treatments, considering patient needs or risks, may rely on insights targeting, for example, pathological signaling cascades of cellular or cellular response dysfunction. Medical protein chemistry is a broad field, employing physics, chemistry, biology, bioinformatics, and medical technology to analyze and explain protein structure and mechanisms, identify underlying molecular genetic abnormalities in disease, and develop interventions to correct them. This perspective emphasizes cellular and / or molecular phenomena and involvement, rather than the previous conceptual and observational focus on the patient and their organs. Protein-based treatments require appropriate protein and treatment recommendations to identify patients for whom a particular therapeutic measure would be beneficial. Therefore, treatment development heavily relies on the investigation of new molecularly targeted drugs and techniques.

[0003] Ischemia-reperfusion injury (IRI) is often involved in a wide range of pathologies, including ischemic stroke, myocardial infarction, acute kidney injury, cardiovascular surgery, and organ transplantation. When blood supply returns to tissues after ischemia, this reoxygenation load damages the tissues. Paradoxically, this necessary reoxygenation load exacerbates cellular dysfunction and apoptosis after restoring blood flow to the previously ischemic tissue. IRI can occur in a wide range of organs, including the heart, lungs, kidneys, intestines, skeletal muscle, and brain, and can not only involve the ischemic organ itself but can also induce systemic damage to distal organs, potentially leading to multi-system organ failure. Reperfusion injury is a multifactorial process that results in widespread tissue destruction. First, insufficient blood flow leads to a deficiency of oxygen, glucose, and other substances required for normal mitochondrial metabolism, including a decrease in ATP levels. This lack of ATP production leads to the cellular ATP-dependent ion pump (Na). + / K + and Ca 2+ The mitochondrial pumps (including the ion pumps) fail, and the transmembrane ion gradient is lost. Damaged or dead cells undergo calcium overload, characterized by calcium accumulation in mitochondria or apoptotic bodies, respectively. The accompanying decrease in ATP (mitochondrial dysfunction) and pyrophosphate levels (a key calcification inhibitor) increases the calcification tendency of these organelles. Similarly, a known complication of organ transplantation that can result from IRI is delayed graft function (DGF). DGF explains the event in which transplant recipients experience poor graft function during the first few weeks after transplantation and require dialysis.

[0004] Therapeutic modalities that make organs more resistant to damage caused by ischemia and reperfusion have yielded controversial results or have only recently emerged. Treatment options that either increase organ resistance to IRI or rescue organs after sudden oxygen restriction have been the focus of major research efforts over the past few decades. Such strategies include, for example, ischemic preconditioning, an experimental strategy in which exposure to a short, non-fatal ischemic episode results in reduced tissue damage during subsequent IRI; metabolic strategies to enhance ischemic tolerance (such as glycolytic enzymes and erythropoietin under the regulation 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 modalities have yielded controversial results or have only recently emerged. Therefore, there remains a significant and unmet need in the treatment of renal impairment, including but not limited to IRI. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, there is a need to provide agents for treating renal impairment, and the present invention provides a direct method for addressing renal impairment, including ischemia-reperfusion injury, in various settings. [Means for solving the problem]

[0006] Fetuin A, a naturally occurring plasma glycoprotein also known as alpha-2-HS-glycoprotein (AHSG), is encoded by the AHSG gene and is derived from the liver. Fetuin A has been reported in the art to attenuate early ischemic brain injury in a cerebral context, with reduced IRI-driven inflammation in rats with ischemic brain tissue (see, e.g., Wang et al., J Cereb Blood Flow Metab. 2010;30(3):493~504). Fetuin A is further associated with the prevention of brain injury in stroke (WO 00 / 60943). However, elevated plasma levels of fetuin A have also been found to increase the risk of myocardial infarction and ischemic stroke, indicating that the role of fetuin A as a prophylactic agent in ischemic brain injury is not well understood (Weickert et al., Circulation. 2008;118:2555~2562).

[0007] Conflicting data have also been presented regarding the use of fetuin A as a biomarker for renal impairment. For example, recent studies investigating the role of fetuin A as a biomarker in kidney transplantation have failed to provide predictive information about patient clinical outcomes (see, e.g., 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 the aforementioned publications, fetuin A levels were generally measured in peripheral blood, which does not reflect fetuin A levels in tissues. Taken together, these publications suggest that fetuin A levels in the blood are not a useful biomarker for long-term survival or clinical prognosis in patients with renal impairment.

[0008] However, in the context of the present invention, it was surprisingly found that administration of fetuin A attenuates renal tissue damage induced by renal hypoxia.

[0009] Accordingly, in a first embodiment, the present invention relates to fetuin A (AHSG) for use in a method for treating renal impairment, wherein an amount of fetuin A effective for treating renal impairment is administered to a subject in need thereof. Preferably, the renal impairment is caused by hypoxia, for example, hypoxia during transplantation, cardiovascular surgery or other major surgery. The renal impairment may be ischemic nephropathy.

[0010] While not bound by any particular theory, based on the experimental evidence provided herein, it is believed that when fetuin A is administered to subjects in need, particularly those suffering from ischemic nephropathy, fetuin A acts as a "mineral scavenger" in hypoxic renal tissue by "removing" calcium levels. Fetuin A is thought to antagonize ectopic calcification, maintain the integrity of renal tissue, and prevent downstream inflammatory cascades that can lead to renal fibrosis through IRI-induced collagen expression. The explanation proposed herein is that fetuin A acts as a "mineral chaperone" in renal impairment, protecting the organ from ischemia-reperfusion injury in the kidney and mitigating harmful inflammatory responses by removing calcium-containing particles.

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

[0012] In preferred embodiments of the present invention, renal injury is selected from the group consisting of acute kidney injury, chronic kidney injury, renal fibrosis, chronic kidney disease (CKD), renal failure, nephritis, acute kidney injury (AKI), ischemic kidney injury, injury related to renal hypoxia, renal ischemia-reperfusion injury (IRI), renal tissue injury, injury related to kidney transplantation, injury related to cardiovascular surgery, and combinations thereof, with renal tissue injury being ischemic renal tissue injury in particular. Injuries related to cardiovascular surgery may be, for example, renal tissue injury resulting from cardiovascular surgery, particularly due to hypoxia and / or ischemia during cardiovascular surgery.

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

[0014] As used herein, “acute kidney injury” refers to a sudden loss or decline in kidney function. This is induced by acute accidental kidney injury or acute kidney injury. There are many causes, including, but are not limited to, prerenal, postrenal, and endogenous acute renal failure. Acute renal failure can also be induced by systemic diseases (such as signs of autoimmune diseases), crush injuries, contrast agents, and certain antibiotics.

[0015] Chronic kidney disease (CKD), chronic nephritis such as chronic renal failure or chronic pyelonephritis, and other “chronic kidney disorders” are also included in the possible treatment spectrum, but are not limited to these. “Chronic kidney disorders” are induced by anatomical location and include, but are not limited to, vascular diseases including macrovascular diseases and ischemic nephropathy such as bilateral renal artery stenosis, small vessel diseases such as hemolytic uremic syndrome and vasculitis, glomerular diseases, tubulointerstitial diseases, obstructive kidney injury, congenital diseases such as polycystic kidney disease, or mesoamerican nephropathy.

[0016] "Fibrosis" refers to the formation of excessive fibrous connective tissue, for example, in the kidney or renal tissue. Tissue inflammation can induce fibrosis. In some embodiments, fibrosis refers to interstitial fibrosis.

[0017] As used herein, “ischemia” or “ischemic” refers to an inadequate blood supply to an organ or a particular part of the body. This is generally considered a serious clinical problem. Ischemia involves a reduced blood supply, and therefore a reduced supply of oxygen and nutrients, as well as a decreased removal of waste products from tissues. This can result in irreversible tissue damage and cell death. Ischemia may occur in the context of multiple kidney disorders, and can be a cause of these. Ischemia may also occur in the context of major surgery; for example, ischemia can result from major surgery. “Reperfusion” is defined herein as the restoration of blood supply after an ischemic event. Reperfusion can cause tissue damage, for example, in the kidneys, particularly by the accumulation of calcium, and above all by an imbalance in mineral balance. “Ischemia-reperfusion injury” (IRI), sometimes called reperfusion injury (RI) or reoxygenation injury, is tissue damage that occurs when blood supply returns (reperfusion) to a tissue after an ischemic period and / or a period of reduced oxygen supply (hypoxia).

[0018] As used herein, “cardiovascular surgery” refers to surgical procedures on major blood vessels and organs in the chest or abdomen, such as surgeries involving the heart, major blood vessels, or lungs. In some embodiments, cardiovascular surgery may be coronary artery bypass surgery or abdominal aortic aneurysm surgery. Abdominal aortic aneurysm surgery can cause renal ischemia-reperfusion injury. Even if cardiovascular surgery is not performed directly on the kidney, renal damage may be associated with it. Ischemia during cardiovascular surgery can cause and may be a cause of renal tissue damage. The occurrence of acute kidney injury (AKI) after cardiovascular surgery is well known, and some embodiments relate to the treatment of acute kidney injury resulting from cardiovascular surgery.

[0019] In the context of the present invention, “subjects requiring it” generally refers to any person who is expected to have renal impairment, is at risk of developing renal impairment, or already has renal impairment, particularly renal impairment, preferably ischemic renal impairment, and has already been diagnosed with it. Subjects may also be referred to as “patients.” Subjects may require prophylactic or therapeutic treatment. The expectation of an increased risk of developing renal impairment may, in some embodiments, be due to a planned major surgery that is at risk of causing ischemia, or even more so, is expected, for example, an increased risk of renal impairment due to a planned kidney transplant or cardiovascular surgery. In some embodiments, subjects requiring it are expected to undergo or have undergone major surgery, preferably a kidney transplant or cardiovascular surgery, or have undergone such surgery.

[0020] As used herein, “treatment,” “to treat,” or “treating” encompasses therapeutic and prophylactic treatments (prevention) of a disorder. “Treatment,” “to treat,” or “treating” may include administering a protein or compound described herein to thereby (i) reduce or eliminate at least one symptom of a particular disorder, or (ii) slow the progression of the disorder, or (iii) stop or prevent the onset of at least one symptom of a particular disorder. As used herein, “therapeutic treatment” and “therapeutic treatment” refer to treatment of an existing disorder, i.e., treatment after the onset of the disorder, whose purpose is to eliminate, mitigate, or slow the existing disorder or reduce its severity. In contrast, as used herein, “prophylactic treatment,” “prevention,” “prevention,” or “prevention” refer to a type of treatment of a disorder intended to reduce risk, preventing or delaying the development of the disorder or its symptoms and / or signs in a subject who does not currently have the disorder, in the future, particularly after an event such as a major surgery like a transplant. In a preferred embodiment, therapeutic treatment means improving the prognosis of the disorder. In some embodiments, prophylactic measures are taken before, during, and / or after major surgery such as kidney transplantation or cardiovascular surgery to reduce the risk of developing complications associated with major surgery, particularly those related to ischemia during kidney transplantation or cardiovascular surgery. In some further embodiments, the terms “treating” or “treatment” preferably mean that the symptoms of the complication are alleviated, but the complication itself is improved, delayed, or prevented, in particular by a reduction in the severity of the complication or by prevention of the complication. Furthermore, “treatment” and “treating” may include the administration or application of a therapeutic agent to a subject or patient, or the performance of a procedure or modality on a subject to obtain a therapeutic or preventive benefit in relation to the complication. For example, treatment may include the administration of a pharmaceutically effective amount of fetuin A. In some embodiments, the complication described above is a kidney disease.

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

[0022] In an alternative embodiment, the present invention relates to fetuin A for use in a method for its prevention, also referred to as prophylactic treatment of a renal disorder, wherein an amount of fetuin A effective to prevent the renal disorder is administered to a subject who needs it, particularly a subject at risk of suffering a future disorder, such as a subject scheduled for a major surgery that may involve renal ischemia. In said alternative embodiment, "treating" or "treatment" means "preventing" or "prevention" of a renal disorder before the onset of said disorder. In some cases, at least one dose of fetuin A is administered within 48 hours before a surgery involving ischemia, particularly a transplantation surgery.

[0023] In another embodiment of the present invention, the renal disorder is related to kidney transplantation, particularly a disorder caused thereby, and particularly, said disorder is selected from the group consisting of delayed graft function (DGF), organ rejection of a kidney graft, kidney tissue injury caused by kidney transplantation, inflammation caused by kidney transplantation, ischemia-reperfusion injury (IRI) caused by kidney transplantation, and combinations thereof. In some embodiments of the present invention, the renal disorder is a disorder caused by kidney transplantation, and said disorder is selected from the group consisting of delayed graft function, kidney tissue injury caused by kidney transplantation, ischemia-reperfusion injury caused by kidney transplantation, and combinations thereof.

[0024] In some embodiments, the renal disorder is an ischemic renal disorder, particularly an ischemic renal disorder caused by kidney transplantation or cardiovascular surgery.

[0025] In another preferred embodiment of the present invention, kidney disorders, particularly ischemic kidney disorders, are related or due to hypoxia, and in particular, the hypoxia is due to ischemia during major surgery, such as ischemia during cardiovascular surgery or kidney transplantation.

[0026] As used herein, "major surgery" is defined by invasive procedures, particularly invasive procedures where resection is performed. Major surgery can include entering the body cavity, removing organs, transplanting organs and / or altering the normal anatomical structure. In some embodiments, major surgery includes local or general anesthesia of the subject. Generally, when opening the mesenchymal barriers (pleural cavity, peritoneum, meninges), the surgery is considered "major" surgery. For example, both kidney transplantation and cardiovascular surgery are major surgeries. When the term "surgery" is used without specifying that it is major surgery, this also includes embodiments of "major surgery" in accordance with the present invention.

[0027] In the context of the present invention, "kidney tissue damage" includes all forms of tissue damage that are incidentally caused, particularly those resulting from hypoxia during kidney transplantation, cardiovascular surgery or other major surgeries, in all cases of hypoxia.

[0028] As used herein, "hypoxia" refers to a state in which the appropriate oxygen supply is depleted at the tissue level in the body or a body region. This is often part of a pathological disorder. Hypoxia-induced tissue damage includes all disorders in which the oxygen supply is lost or reduced, causing oxygen supply deficiency in any tissue. This also generally includes a reduction in blood supply due to major surgery or vascular injury. The term "hypoxia" also includes anoxia, i.e., complete depletion of oxygen levels, as an extreme form of hypoxia.

[0029] In another preferred embodiment of the present invention, IRI is due to hypoxia during surgery, particularly major surgery. Hypoxic disorders caused during such surgery, particularly major surgery, can result in ischemic reperfusion injury in the kidney, thereby damaging the tissue. Ischemic reperfusion injury can be due, at least in part, to calcification.

[0030] In a further embodiment of the present invention, the renal impairment is graft function delay, renal fibrosis, or a combination thereof.

[0031] As used herein, “fetuin A” refers to either naturally occurring fetuin A, or a protein or fragment or derivative thereof that does not exist naturally but has the same or similar function as naturally occurring fetuin A, in particular the same or similar function as naturally occurring human fetuin A. Preferably, it is a calcification inhibitor and / or its protein sequence is at least partially identical to that of human fetuin A. In some embodiments, fetuin A is plasma-derived or recombinant human fetuin A. Human plasma-derived fetuin A is obtained from human plasma. The term fetuin A also encompasses all naturally occurring alleles, splice variants and isoforms, as well as synthetic peptides, peptidomimetic or peptide fragments, that have the same or similar function as naturally occurring fetuin A and / or human fetuin A, such as inhibition of calcification.

[0032] In some embodiments of the present invention, fetuin A is human fetuin A. In some embodiments, human fetuin A is derived from human plasma, or alternatively, human fetuin A is recombinant human fetuin A. Plasma-derived human fetuin A is purified from human plasma. Recombinant human fetuin A may have a different glycosylation pattern than plasma-derived human fetuin A. In some embodiments, fetuin A is naturally occurring fetuin A. As referred to in the context of the present invention, “naturally occurring protein” generally refers to a protein that is found naturally in any type of living organism and can be isolated as such from the tissues, fluids, and / or any individual cells of said organism. For example, some naturally occurring proteins can be obtained from human plasma. Recombinant-produced proteins also include fetuin A derivatives that are marked or labeled in some way by conjugating a gene tag (such as a FLAG-tag, His-tag, Myc-tag, HA-tag, or a fluorescently labeled protein such as GFP, YFP, or RFP), which may or may not facilitate purification.

[0033] As used herein, “purified” generally refers to the purification of proteins, which are well known to those skilled in the art. Generally, proteins are purified based on their properties, such as solubility, size, charge, and specific binding affinity. That is, proteins are often separated by ion-exchange chromatography based on their net charge. Protein purification techniques are well known to those skilled in the art. These techniques include, at one level, homogenization of cells, tissues, or organs, and fractionation of these into polypeptide and non-polypeptide fractions. Unless otherwise specified, the protein or polypeptide of interest may be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity). Methods particularly suitable for the production of pure peptides include ion-exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, hydrophobic interaction chromatography, immunoaffinity chromatography, and isoelectric focusing. High-performance liquid chromatography is a method for purifying peptides. Another method for protein purification is high-performance protein liquid chromatography. For example, the purification of plasma proteins can be carried out by plasma fractionation and / or chromatography steps. Protein purification may also include virus inactivation as a process. The term "purified" does not rule out the presence of some impurities, but generally, other blood components and / or impurities are primarily removed.

[0034] In another preferred embodiment of the present invention, a functionally active derivative or fragment of human fetuin 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 Sequence ID No. 1 as defined below. “Homologous” is defined herein as sequence similarity of various biological sequences at the RNA, DNA, and protein sequence levels. “Functionally active derivative” has the same or similar activity as naturally occurring fetuin A and may inhibit calcification in particular. The percentage of sequence identity can be determined, for example, by sequence alignment. Methods for sequence alignment 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 from the internet for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0035] Accordingly, in another embodiment of the present invention, fetuin A comprises or consists of the amino acid sequence or one or more fragments thereof according to SEQ ID NO: 1. In the context of the present invention, the terms “one or more fragments thereof” refer to any amino acid sequence or peptide as defined by SEQ ID NO: 1, including but not limited to any small fragment or fragment containing additional amino acids that exhibit fetuin A activity, as defined above for fragments and derivatives of naturally occurring proteins having fetuin A activity. 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, where the length is related 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. The protein fragments of the present invention can be obtained by altering the protein sequence (e.g., by the deletion, substitution, and / or addition of one or more amino acids), and the altered protein retains the function of the unaltered protein, i.e., its ability to localize to an intracellular structure selected from the group consisting of the cell surface, contractile ring, and centrosome during the progression of the cell cycle, or to function as a fluorescent reporter protein. Such sequence alterations may include, but are not limited to, conservative substitutions, deletions, mutations, and / or insertions.

[0036] Human fetuin A sequences (protein and nucleic acid sequences) are available from the National Center for Biotechnology Information (NCBI).

[0037] Sequence ID 1 (protein sequence of human fetuin A) MKSLVLLLCLAQLWGCHSAPHGPGLIYRQPNCDDPETEEAALVAIDYINQNLPWGYKHTLNQIDEVKVWPQQPSGELFEIEIDTLETTCHVLDPTPVARCSVRQLKEHAVEGDCDFQLLKLDGKFSVVYAKCDSSPADSAEDVRKVCQDCPLLAPLNDTRVVHAAKAALAAFNAQNNGSNFQLE EISRAQLVPLPPSTYVEFTVSGTDCVAKEATEAAKCNLLAEKQYGFCKATLSEKLGGAEVAVTCMVFQTQPVSSQPQPEGANEAVPTPVVDPDAPPSPPLGAPGLPPAGSPPDSHVLLAAPPGHQLHRAHYDLRHTFMGVVSLGSPSGEVSHPRKTRTVVQPSVGAAAGPVVPPCPGRIRHFKV Protein sequence: NP_001341500.1 (368th amino acid) (https: / / www.ncbi.nlm.nih.gov / protein / NP_001341500.1)

[0038] Sequence ID 2 (Nucleic acid sequence of human fetuin A) Nucleic acid sequence: NM_001354571.2 (nucleotide at position 1107) (https: / / www.ncbi.nlm.nih.gov / gene / 197;NM_001354571.2;Consensus coding sequence CCDS87176.1)

[0039] In another preferred embodiment, a functionally active derivative or fragment of the naturally occurring protein fetuin 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 Sequence ID No. 3 (bovine fetuin A sequence) as defined below.

[0040] Sequence ID 3 (protein sequence of bovine fetuin A) MKSFVLLFCLAQLWGCHSIPLDPVAGYKEPACDDPDTEQAALAAVDYINKHLPRGYKHTLNQIDSVKVWPRRPTGEVYDIEIDTLETTCHVLDPTPLANCSVRQQTQHAVEGDCDIHVLKQDGQFSVLFTKCDSSPDSAEDVRKLCPDCPLLAPLNDSRVVHAVEVALATFNAESNGSY LQLVEISRAQFVPLPVSVSVEFAVAATDCIAKEVVDPTKCNLLAEKQYGFCKGSVIQKALGGEDVRVTCTLFQTQPVIPQPQPDGAEAEAPSAVPDAAGPTPSAAGPPVASVVVGPSVVAVPLPLHRAHYDLRHTFSGVASVESSSGEAFHVGKTPIVGQPSIPGGPVRLCPGRIRYFKI Protein sequence: NP_776409.1 (359th amino acid) (https: / / www.ncbi.nlm.nih.gov / protein / NP_776409.1)

[0041] Sequence ID 4 (nucleic acid sequence of bovine fetuin A) Nucleotide sequence: NM_173984.3 (encoding nucleotide sequence (1080th nucleotide)) (https: / / www.ncbi.nlm.nih.gov / nuccore / NM_173984.3)

[0042] In another embodiment of the present invention, fetuin A is recombinantly expressed in a host cell line, preferably to provide post-translational modification, more preferably glycosylation.

[0043] As used herein, the term “expressed” generally means being able to make information in a gene or DNA sequence apparent or making it apparent. In particular, in the context of the present invention, it means intracellular production of a protein encoded by the nucleic acid expression construct of the present 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 a cell to form an “expression product,” such as a protein. Protein expression in or by a cell may depend on a variety of factors, including, but not limited to, a promoter sequence operably linked to the DNA sequence encoding the protein of interest, the presence or absence of enhancer and / or silencer sequences or other DNA sequences that control the transcription rate, cell culture conditions applied to cells having each DNA sequence, including the medium used to culture the cells, and / or the copy number of the DNA sequence introduced into the cell. The success of fusion protein expression can be analyzed and / or visualized by standard methods known to those skilled in the art, including, but not limited to, methods involving Western blotting, Northern blotting, RNase protection assays, quantitative RT-PCR, and direct or indirect fluorescence readout. "Recombinant" and "recombinantly" mean artificially produced and relate to or indicate organisms, cells, proteins, or genetic material formed by recombination. Recombinant expression of a desired protein is a standard method in the art, followed by the standard protein purification methods described below.

[0044] Post-translational modifications enhance the functional diversity of the proteome through the covalent addition of functional groups or proteins, proteolytic cleavage of regulatory subunits, or degradation of the entire protein. These modifications include, in particular, phosphorylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, lipidation, and glycosylation.

[0045] A "host cell that provides post-translational modification" is a host cell that delivers the necessary modification modalities, enabling precise post-translational modification of mammalian proteins. Systems that provide these requirements include, for example, insect cell expression systems such as BTI-Tn5B1-4 or SF9 cell lines, or mammalian cell expression systems such as HEK293(T), CHO, or HELA cells, but are not limited to these.

[0046] Glycosylation is defined as an enzyme-driven, site-directed process that links sugars to produce glycans (which are then bound to proteins, lipids, or other organic molecules). As a post-translational process, protein glycosylation is a natural cellular process in which sugars are selectively attached to specific protein residues to confer high structural stability or function to the native protein structure. In this regard, glycosylation plays a crucial role in the proper functionality of a variety of proteins. Examples of glycosylation include, but are not limited to, C-linking, N-linking, O-linking, or S-linking glycosylation and glycation. In this context, glycation refers to the non-enzymatic binding of reducing sugars to the nitrogen atoms of proteins.

[0047] As used herein, “injection” refers to parenteral mode of administration, not including oral administration or inhalation. As used herein, “injection” includes intravenous (iv), intra-arterial, intramuscular (im), subcutaneous (sc) and intradermal (id), intraperitoneal, intraosseous, intracardiac, intra-articular and intracavitary administration. In some advantageous embodiments, injection refers to intravenous (iv) or subcutaneous (sc) administration. Injection may be a discontinuous and / or short-duration injection using a needle with, for example, a single dose of fetuin A (and optionally subsequent injections of further doses). Alternatively, injection may also be an infusion, i.e., a long-duration continuous injection. In some embodiments, 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 administration by extracorporeal circulation (ECC). As used herein, “inhalation” describes a method of inhalation or breathing that can deliver air, gases, active substances and aerosols to the lungs. For example, fetuin A is provided as part of an aerosol for the purpose of inhalation.

[0048] In some embodiments of the present invention, fetuin A is administered to the patient by injection or inhalation. In some preferred embodiments, fetuin A is administered to the patient by injection. In some embodiments, fetuin A is administered to the patient by intravenous (iv), intramuscular (im), or subcutaneous (sc) injection, preferably intravenous (iv) injection.

[0049] In some embodiments, fetuin A is administered to the patient in an amount effective to treat renal impairment, particularly fetuin A is administered to the patient at concentrations in the range of 1 mg / kg to 200 mg / kg body weight, particularly intravenously. In some embodiments, fetuin A is administered to the patient at concentrations in the range of 5 mg / kg to about 100 mg / kg body weight, particularly intravenously. In some embodiments, fetuin A is administered to the patient at concentrations in the range of 10 mg / kg to about 50 mg / kg body weight, particularly intravenously. In some embodiments, fetuin A is administered to the patient at concentrations in the range of 10 mg / kg to about 75 mg / kg body weight, particularly intravenously. In some embodiments, fetuin A is administered to the patient at concentrations in the range of 20 or 30 mg / kg to about 200 mg / kg body weight, particularly intravenously. In some embodiments, fetuin A is administered to the patient at concentrations in the range of 20 mg / kg to about 50 mg / kg body weight, particularly intravenously. In some embodiments, fetuin A is administered to patients at concentrations ranging from 30 mg / kg to approximately 50 mg / kg body weight, particularly intravenously.

[0050] In further embodiments, fetuin A is administered to subjects who require it during major surgery, for example, during cardiovascular surgery or kidney transplantation. In further embodiments, fetuin A is administered to subjects who require it after major surgery, for example, after cardiovascular surgery or kidney transplantation. In further embodiments, fetuin A is administered to subjects who require it before major surgery, for example, before cardiovascular surgery or kidney transplantation. These embodiments can be combined, for example, in some embodiments, fetuin A is administered before and after major surgery.

[0051] In the context of the present invention, fetuin A may be a target of the transcription factor HIF-1α. HIF-1α, also known as hypoxia-inducible factor 1-alpha, 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 cellular signaling pathway or cascade. In the context of the present invention, fetuin A may act downstream of HIF-1α.

[0052] In some embodiments of the present invention, the treatment includes regulating the level of calcification in kidney tissue, preferably a level of hypoxia-related calcification. "Calcification" is defined herein as the accumulation of calcium salts in body tissue, particularly in the kidney, which can result in permanent tissue damage. This can lead to an inflammatory response and tissue damage as a result of restricted oxygen supply to the tissue. In one embodiment, the treatment includes the removal of calcium mineral deposits in kidney tissue, particularly in transplanted kidney tissue.

[0053] In another embodiment of the present invention, at least two doses of fetuin A are administered, or at least three doses of fetuin A are administered. In some embodiments, at least four doses of fetuin A are administered. In another embodiment of the present invention, the treatment in question preferably includes a treatment period of at least one dose of fetuin A administered at least daily or weekly during the week or month following an ischemic accident and / or surgery, particularly major surgery.

[0054] In the context of this invention, the term “ischemic accident” refers to any event that causes and / or involves ischemia, in particular ischemia. Surgery is an ischemic accident in which the blood supply to tissue is restricted during the surgery. For example, during a kidney transplant procedure, the graft may often not be adequately perfused with blood. Sudden blood loss during an accident may also be an ischemic accident, during which the kidney is not adequately perfused. In some embodiments, an ischemic accident is ischemia during a major surgery, in particular, where the major surgery is a kidney transplant or cardiovascular surgery.

[0055] In another embodiment of the present invention, at least one dose of fetuin A is administered before, during, or after an ischemic accident and / or surgery, particularly major surgery. In some embodiments, the ischemic accident and / or surgery is a kidney transplant or cardiovascular surgery.

[0056] In yet another embodiment of the present invention, at least one dose of fetuin A is administered before or within 48 hours thereafter, particularly within 24 hours, of an ischemic event such as a kidney transplant or cardiovascular surgery. In some embodiments, at least one dose of fetuin A is administered within 48 hours after an ischemic event, particularly within 24 hours. In yet another embodiment, at least one dose of fetuin A is administered within 48 hours before a major surgery, particularly within 24 hours. In yet another embodiment, at least one dose of fetuin A is administered within 48 hours after a major surgery, particularly within 24 hours. In some further embodiments, fetuin A is administered during a major surgery, which in some embodiments is a kidney transplant or cardiovascular surgery.

[0057] In yet another embodiment of the present invention, at least one first dose of fetuin A is administered within 48 hours prior to an ischemic event, particularly within 24 hours; and at least one second dose of fetuin A is administered within 48 hours prior to an ischemic event, particularly within 24 hours. In some embodiments, at least two doses of fetuin A are administered within 48 hours prior to an ischemic event, particularly within 24 hours. In yet another embodiment, at least two doses of fetuin A are administered within 48 hours prior to a major surgery, particularly within 24 hours. In yet another embodiment, two or three doses of fetuin A are administered within 48 hours prior to a major surgery, particularly within 24 hours. In some embodiments, at least two doses of fetuin A are administered within 48 hours prior to an ischemic event, particularly within 24 hours. In yet another embodiment, at least two doses of fetuin A are administered within 48 hours prior to a major surgery, particularly within 24 hours. In yet another embodiment, two or three doses of fetuin A are administered within 48 hours prior to a major surgery, particularly within 24 hours.

[0058] In some embodiments, fetuin A is administered to the patient at least once a week, particularly at least once every 48 hours, preferably at least once every 24 hours.

[0059] In some embodiments, fetuin A is administered in divided doses over a total duration of 1 to 100 days, particularly over a total duration of 1 to 50 days. In other embodiments, fetuin A is administered in divided doses over a total duration of 1 to 25 days. In another embodiment, fetuin A is administered in divided doses over a total duration of 1 to 10 days. In yet another embodiment, fetuin A is administered in divided doses over a total duration of 2 to 10 days.

[0060] In embodiments of the present invention, the treatment involves administering fetuin A as part of a pharmaceutical composition to a subject requiring it. Preferably, the pharmaceutical composition is in liquid form. Preferably, the pharmaceutical composition is one or more of the embodiments defined below in a second embodiment of the present invention.

[0061] In a second embodiment, the present invention relates to a pharmaceutical composition for use in the treatment of renal impairment, comprising fetuin A (AHSG) and optionally at least one pharmaceutically acceptable carrier. In some embodiments, the second aspect relates to a pharmaceutical composition for use in a method for treating renal impairment, wherein an amount of fetuin A effective for treating renal impairment is administered to a subject, preferably a patient, that requires it.

[0062] The term "pharmaceutical carrier" as used herein may be any substrate used in a drug delivery method that helps improve the selectivity, efficacy, and / or safety of drug administration.

[0063] The pharmaceutical composition according to the second embodiment can be used for the same treatments and renal disorders described above in relation to fetuin A, and any treatment described above may also illustrate embodiments of the pharmaceutical composition for use in such treatments. In a preferred embodiment of this second embodiment, the renal disorder is selected from the group consisting of acute kidney injury, chronic kidney injury, renal fibrosis, chronic kidney disease, renal failure, nephritis, acute kidney injury, ischemic kidney injury, hypoxia-related injury, renal ischemia-reperfusion injury, renal tissue injury, preferably ischemic kidney tissue injury, kidney transplant-related injury, cardiovascular surgery-related injury, and combinations thereof.

[0064] In some embodiments, the pharmaceutical composition is a liquid. In some embodiments, the pharmaceutical composition contains water.

[0065] In another embodiment of this second aspect, the kidney transplant-related disorders are selected from the group consisting of delayed graft function, organ rejection of the kidney graft, kidney tissue damage resulting from kidney transplantation, inflammation resulting from kidney transplantation, renal IRI resulting from kidney transplantation, and combinations thereof.

[0066] Preferably, the renal impairment is caused by hypoxia during surgery, especially major surgery. For example, hypoxia may result from ischemia during kidney transplantation or cardiovascular surgery.

[0067] Similarly, in a preferred embodiment, fetuin A of the pharmaceutical composition is defined as described in detail above. The pharmaceutical composition may comprise any of the above embodiments of fetuin A.

[0068] In the context of the pharmaceutical composition of the present invention, fetuin A is preferably human fetuin A, and more preferably fetuin A derived from human plasma.

[0069] In an alternative embodiment, fetuin A of the pharmaceutical composition is recombinantly expressed in a host cell line, preferably to provide post-translational modifications, particularly glycosylation.

[0070] It is equally preferable in this context that fetuin 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 preferable that fetuin A includes or consists of SEQ ID NO: 1 or a fragment thereof.

[0071] In the embodiment, the pharmaceutical composition is administered to the subject as described in detail above. Preferably, the pharmaceutical composition is formulated for injection or inhalation.

[0072] The following drawings and embodiments are intended to illustrate various embodiments of the present invention. Therefore, specific modifications considered herein should not be understood as limitations of the scope of the invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the invention, and therefore, it should be understood that such equivalent embodiments should be included herein. [Brief explanation of the drawing]

[0073] [Figure 1-1] Chronic fetal hypoxia induces restricted intrauterine growth in mice: Figure 1A. Experimental setup and time of analysis. Figure 1B. Mean number of offspring per litter. Figure 1C. Total fetal weight distribution (n=49 for each experimental condition; No: normal oxygen, Hy: hypoxia, Cc: calorie restriction). Figure 1D. Mean nephron number per kidney was determined by staining for glomerular marker nephrin. Independent t-test with Welch's correction. [Figure 1-2] Continuation of Figure 1-1. [Figure 2-1]Hypoxia-inducible gene expression in the kidney: Figure 2A. Hierarchical clustering of cDNA microarray data comparing renal gene expression profiles in hypoxia, normoxic, and calorie-restricted E18.5 fetuses (n=3 for each experimental condition). White areas indicate induction, and vertical hatched areas indicate suppression (the degree of induction and suppression is not shown in Figure 2A). The location of Ahsg is indicated by an arrow. Gene clustering was performed with at least 1.3-fold regulation of hypoxia compared to both normoxic controls; one-way ANOVA; P<0.05. Figures 2B-E. Relative mRNA levels of Ahsg (B), Apoa2 (C), Fgg (D), and Gys2 (E) in E18.5 kidney (circle) or liver (square) samples. Figure 2F. Fetuin A plasma levels of E18.5 fetuses as assessed by ELISA. [Figure 2-2] Continuation of Figure 2-1. [Figure 2-3] Continuation of Figure 2-2. [Figure 2-4] Continuation of Figure 2-3. [Figure 3] Fetal hypoxia-induced fetuin A expression in the proximal tubule: Figure 3A-H. Fetuin A staining in E18.5 kidney sections of normal-oxygen (A-D) or hypoxic (E-H), wild-type (A, C, E, G) or Clcn5 KO (B, D, F, H) fetuses. Arrowheads (D, H) indicate intraluminal fetuin A staining resulting from impaired endocytosis of low molecular weight proteins in the PT of Clcn5 KO mice. (I-L) Immunofluorescence staining of nephron segment marker proteins and fetuin A shown in E18.5 kidney sections. PT, proximal tubule; TAL, thick ascending limb; DCT, distal curved tubule; CD, collecting duct; scale bar, 300 μm (overall image) or 50 μm. [Figure 4-1]In vitro hypoxia-activated fetuin A expression: Figure 4A. Depiction of potential HRE in mouse Ahsg used to produce luciferase reporter gene constructs (1-8). Mutant HRE is shown. Figure 4B. Luciferase activity in NRK cells transfected with reporter constructs. The fold change in luciferase luminescence between hypoxic and normoxic culture conditions is shown. Each condition is normalized to an empty vector control (pGL3). Dunnett multiple comparison test. Figures 4C-E. Fetuin A expression in rat cell line NRK (D) and human cell line HK-2 (E) in mouse primary proximal tubular cells (pPTC) (C) isolated from four different mice cultured under normoxic or hypoxic conditions. [Figure 4-2] Continuation of Figure 4-1. [Figure 5-1] Fetuin A deficiency exacerbated CKD progression in hypoxic IUGR kidneys: Figure 5A-B. Reduced renal function in 9-week-old mice, assessed by protein / creatinine ratio and glomerular filtration rate (GFR). Figure 5B. Most pronounced in fetal hypoxia-Ahsg KO animals, showing an additive effect of hypoxia and fetuin A deficiency (n≧5 for each experimental condition). Figure 5C-D. Representative images of picrosilius red staining of collagen show stronger and more complex patterns in kidney sections from P14 fetal hypoxia-Ahsg KO mice (D) compared to fetal hypoxia-wt mice (C). Figure 5E-G. Relative expression levels of fibrosis markers Acta2 (E), Col1a1 (F), and Fn1 (G) are significantly enhanced in the kidneys of fetal hypoxia-Ahsg KO mice (n≧5 for each experimental condition). Fisher's LSD test (A-B). Scale bar, 50 μm. [Figure 5-2] Continuation of Figure 5-1. [Figure 5-3] Continuation of Figure 5-2. [Figure 6-1]Fetuin A deficiency promoted the accumulation of calcium mineral particles and macrophages in hypoxic IUGR kidneys: Figures 6A-I. Detection of calcium biominerals by ATTO488 fluorescently labeled fetuin A (488-FA) staining in E18.5 kidney sections of normoxic wt (A, D, G), hypoxic wt (B, E, H), or hypoxic Ahsg KO embryos (C, F, I). Hypoxic Ahsg KO mice showed the strongest staining intensity in PT(C) compared to normoxic and hypoxic wt mice (A, B), indicating increased mineralization matrix turnover. Arrowheads point towards the granular staining pattern, reflecting the massive accumulation of 488-FA in the papilla (F) and cortex (I) present only in hypoxic Ahsg KO mice. Figures 6J-M. Representative immunofluorescence staining of macrophage markers F4 / 80 (j', k', l', m') and fetuin A (j''', k''', l''', m'''), counterstaining with DAPI (j'''', k'''', l'''', m'''') and 488-FA (j'', k'', l'', m'') in E18.5 kidney sections under the indicated genotypes and oxygen conditions. Macrophage accumulation and diffusion, as well as granular 488-FA staining, were detected only in hypoxic Ahsg KO mice (M). Figure 6N. Quantification of total F4 / 80 stained area per field of view. Multiple sections from at least three mice were analyzed for each experimental condition. Scale bar, 100 μm. [Figure 6-2] Continuation of Figure 6-1. [Figure 6-3] Continuation of Figure 6-2. [Figure 7-1]Fetuin A attenuated the hypoxia-induced expression of fibrosis markers: Figure 7A. Representative Western blots showing the induction of fibronectin and α-smooth muscle actin (α-SMA) under hypoxia culture conditions in primary proximal tubular cells (pPTCs) isolated from wt or Ahsg KO mice. Figures 7B-D. Fetuin A supplementation attenuates the hypoxia-induced expression of fibrosis markers Acta2 (B), Col1a1 (C), and Fn1 (D) in pPTCs (n≧5 for each experimental condition). Figures 7E-F. Only fetuin A had a reducing effect on the expression of fibrosis markers; BSA did not (n≧4 for each experimental condition). Figure 7G. Representative Western blots showing that fetuin A supplementation attenuates the expression of fibronectin and type I collagen (Col1a1) protein. Figure 7H. TGF-β1 treatment and hypoxia have an additive effect on Smad3 phosphorylation. Representative Western blots are shown in Figure 7I. Quantification of Smad3 activation shown in (H). Independent t-tests with Welch correction (B-D, comparison only of normal oxygen wt and normal oxygen Ahsg KO samples). Fisher's LSD test (I). [Figure 7-2] Continuation of Figure 7-1. [Figure 7-3] Continuation of Figure 7-2. [Figure 7-4] Continuation of Figure 7-3. [Figure 7-5] Continuation of Figure 7-4. [Figure 8] Hypoxic-responsive IRI induced the deposition of calcium-containing microparticles in the kidney: detection of calcium-containing microparticles by 488-FA after ischemia-reperfusion (IRP). 488FA staining in the control side (I) and ischemia-reperfusion (m) 7 days after surgery showed the presence of calcium biominerals only in the IRP kidney. [Figure 9-1] Fetuin A supplementation attenuated the expression of fibrosis markers after ischemia-reperfusion injury: Figures 9A-F. Fetuin A supplementation attenuated the ischemia-induced expression of Col1a1 (A), Col3a1 (B), and Col6a1 (C), but had no effect on the expression of non-collagenary fibrosis markers Acta2 (D), Fn1 (E), or Vim (F). One-way ANOVA test. [Figure 9-2] Continuation of Figure 9-1. [Figure 9-3] Continuation of Figure 9-2. [Figure 9-4] Continuation of Figure 9-3. [Figure 10-1] Prophylactic administration of human fetuin A in mice undergoing ischemia-reperfusion injury (IRI) reduced injury and decreased fibrotic remodeling: Figures 10A-D. Expression levels of early injury markers Kim1(A), Krt18(B), Krt20(C), and Lcn2(D) were decreased in IRI kidneys pre-treated with fetuin A compared to IRI kidneys pre-treated with PBS. Figures 10E and F. Fibrotic remodeling markers Clu(E) and Tgfb1(F) were similarly decreased in IRI compared to PBS controls. Figure 10G. The non-collagen fibrosis marker Vim(G) was decreased in IRI kidneys pre-treated with fetuin A compared to IRI kidneys pre-treated with PBS. Figures 10H-J. Collagen expression during prophylactic fetuin A treatment is shown for Col1a1(H), Col3a1(I), and Col6a1(J). Figure 10K. Arg1(K) expression was suppressed in fetuin A-pretreated mice compared to PBS-pretreated mice, indicating a reduced need for collagen synthesis. Figure 10L-N. Fetuin A pretreatment also reduces the expression of chemokines Ccl2(L), II-6(M), and Tnf-α(N), which are involved in damage signaling and repair that normally increase under ischemic conditions. Legends in Figure 10: White circles: PBS-injected, unsurgical control kidney; White triangles: PBS-injected, IRI kidney; Black circles: Fetuin A-injected, unsurgical control kidney; Black triangles: Fetuin A-injected, IRI kidney. P-values ​​in Figure 10 are shown for standard one-way ANOVA with Tukey correction for multiple comparisons (normal font) and paired t-tests (bold font). [Figure 10-2] Continuation of Figure 10-1. [Figure 10-3] Continuation of Figure 10-2. [Figure 10-4] Continuation of Figure 10-3. [Figure 10-5] Continuation of Figure 10-4. [Figure 10-6] Continuation of Figure 10-5. [Figure 10-7] Continuation of Figure 10-6. [Figure 10-8] Continuation of Figure 10-7. [Figure 11-1]Presence of endogenous fetuin A in mouse kidney tissue after intravenous administration: Figure 11A. Western blot. Lanes A-C: Fetuin A injection, unsurgical control kidney; Lanes D-F: Fetuin A injection, IRI kidney; Lanes G-I: PBS injection, unsurgical control kidney; Lanes J-L: PBS injection, IRI kidney; Lane M: Empty; Lane N: Human fetuin A protein used for injection. Top panel: Western blot for endogenous mouse fetuin A using ab187051 anti-fetuin A antibody (specific to mouse fetuin A) from abcam. Middle panel: Western blot for human fetuin A using sc-133146-HRP anti-fetuin A antibody (specific to human fetuin A) from Santa Cruz. Bottom panel: Rplp0 used as a loading control. Kidney samples from mice injected with human fetuin A protein showed a strong band of human fetuin A (lanes A-F). Fetuin A was not detected in the PBS-injected control sample (lanes G-L). PBS-injected IRI kidneys (lanes J-L) showed the highest abundance of mouse fetuin A among all samples. Human fetuin A was not detected by the mouse-specific anti-fetuin A antibody ab187051 because human fetuin A protein (lane N) did not signal. Figure 11B. ELISA for mouse fetuin-A using the MFTA00 ELISA kit (specific to mouse fetuin-A) from bio-techne. Endogenous mouse fetuin A was elevated only in the PBS-injected IRI kidney compared to all other samples. Figure 11C. ELISA for human fetuin-A using the DFTA00 ELISA kit (specific to human fetuin-A) from bio-techne. Human fetuin A was not detected in kidney samples from PBS-injected mice. Fetuin A-injected IRI kidneys had higher tissue levels of human fetuin A than the unsurgery contralateral (right) kidney. Figures 11D-F. Endogenous fetuin A was present in mouse renal tissue after intravenous administration. Fluorescently labeled fetuin A (Alexa-488 labeled fetuin A) was present in the proximal tubule (PT) 15, 30, and 60 minutes after intravenous injection.Fifteen minutes after injection of Alexa-488-labeled fetuin A, a fine, intermittent stain was detected in the proximal tubule. The intensity of this staining increased at 30 and 60 minutes after injection. G, glomerulus. Legends in Figures 11B and C: White circles: PBS injection, unsurgical control kidney; White triangles: PBS injection, IRI kidney; Black circles: Fetuin A injection, unsurgical control kidney; Black triangles: Fetuin A injection, IRI kidney. P-values ​​in Figures 11B and C are shown for standard one-way ANOVA with Tukey correction for multiple comparisons (normal font) and paired t-tests (bold font). [Figure 11-2] Continuation of Figure 11-1. [Figure 11-3] Continuation of Figure 11-2. [Figure 11-4] Continuation of Figure 11-3. [Figure 12-1]Time course of human fetuin A in mouse serum after intravenous administration: Figure 12A. Lanes A and B: 1 minute after injection; Lanes C and D: 1 hour after injection; Lanes E and F: 1 day after injection; Lanes G and H: 1 week after injection; Lanes I and J: Control PBS injection; Lane K: Human fetuin A protein used for injection. Western blot for human fetuin A using human-specific sc-133146-HRP anti-fetuin A antibody from Santa Cruz. After 1 day, the detectable amount of human fetuin A in serum was decreased compared to 1 minute or 1 hour after injection. After 1 week, human fetuin A was not detectable. Mouse fetuin A was not detected with sc-133146-HRP anti-fetuin A antibody because the PBS-injected control animals did not signal. Figure 12B. Normalized ELISA data using the bio-techne DFTA00 ELISA kit (specific for human fetuin A) showing the decrease in intravenous human fetuin A in mouse serum samples. Compared to the time of injection (1 minute), the amount of circulating human fetuin A decreased by 80% after 1 hour and by 8.5% after 1 day. After 1 week, human fetuin A was no longer detectable, similar to PBS-injected mice (black circles). Figure 12C. Time course of human fetuin A in mouse serum after intravenous administration. Normalized ELISA data showing the decrease in intravenous human fetuin A in mouse serum samples. Compared to the time of injection (1 minute), the amount of circulating human fetuin A decreased by 80% after 1 hour and by 8.5% after 1 day. After 1 week, human fetuin A was no longer detectable. [Figure 12-2] Continuation of Figure 12-1. [Modes for carrying out the invention]

[0074] [Examples] Chronic fetal hypoxia induces IUGR in mice. To model chronic fetal hypoxia, pregnant mice bred in timely conditions were exposed to 10% oxygen from E14.5 to E18.5 (Figure 1A). Despite ad libitum food access, hypoxic mothers (Hy) were observed to have poor appetites, and since calorie restriction itself is a known inducer of IUGR5, a second control group was included in the analysis to rule out the possibility that the findings were due to reduced calorie intake rather than hypoxia. In this calorie-restricted (Cc) group, normoxic mothers were fed an amount of food comparable to the small amount of food consumed by hypoxic mice. While litter size, placental weight, or fetal survival rates were indistinguishable among the three groups, maternal weight gain was significantly reduced in hypoxic mothers (Figure 1B). Importantly, only fetuses from hypoxic mothers exhibited LBW and met the IUGR criteria (Figure 1C), while fetuses from normoxic or Cc groups did not. Compared to controls, hypoxic fetuses had smaller kidneys and significantly fewer nephrons (Figure 1D).

[0075] Hypoxic fetal kidneys adopt liver gene expression patterns. Next, whole-genome expression in fetal hypoxic kidneys was examined using gene arrays. 62 inducers and 28 repressors were identified and compared to both control groups (Figure 2A). Seventeen of the inducers, including the true HIF target genes transferrin, trefoil factor 3, neuritin, alpha-1-antitrypsin (serpina 1d), and alpha-1-antichymotrypsin (serpina 3n), are known to be regulated by hypoxia. Furthermore, over 20% of the inducers were found to be frequently purified from CPPs containing the major calciprotein particle (CPP) components fetuin A (Ahsg), albumin, Apo-A1, and thrombin (F2). Functional annotation clustering of the inducers revealed an enrichment of secretory plasma proteins normally transcribed exclusively in the liver in hypoxic kidneys. Validation of microarray data by RT-qPCR of selected genes revealed more than a 10-fold induction only in hypoxic fetal kidneys, but not in the control group kidneys (Figures 2B-E). No changes in mRNA levels were found in fetal liver samples.

[0076] Fetuin A is produced locally in the proximal tubules under hypoxic conditions. Interestingly, the gene with the highest induction (Ahsg) was found in 7 out of 10 annotated groups. Ahsg belongs to the cystatin superfamily of cysteine ​​protease inhibitors, encodes fetuin A, a negative acute-phase glycoprotein, and its main function is related to the metabolism of the mineralized matrix. Despite its strong induction in hypoxic kidneys, no significant increase in fetal plasma fetuin A levels was detected (Figure 2F), suggesting a specific, systemic rather than systemic, functional relevance during the hypoxic challenge. To further address the functional relevance of Ahsg expression in fetal hypoxic kidneys, its precise localization was investigated. Figure 3 shows immunohistochemistry of the fetal renal proximal tubules (Figure 3A, B, E, F) or tubular lumen (Figure 3C, D, G, H). Strong fetuin A staining defined the cell boundaries of the proximal tubule (PT) regardless of oxygen conditions (Figure 3A, E). This is due to systemic fetuin A filtration and its uptake into the PT via megalin-dependent endocytosis, which shields any fetuin A locally produced in hypoxic fetal kidneys. To selectively visualize renal fetuin A protein, we employed a genetic approach that blocks endocytosis to the PT, an alternative to pharmacological inhibition of megalin-dependent endocytosis using His-sRAP (histidine-tagged soluble receptor-associated protein). Clcn5 knockout (KO) mice exhibited severe endocytotic impairment of low molecular weight proteins in the PT, mimicking Dent's disease. In normoxic Clcn5 KO kidneys, there was no marked fetuin A staining in PT cells (Figure 3B). Instead, strong intraluminal signals were detected (Figure 3D), which were not present in wild-type (wt) samples (Figure 3C), highlighting the endocytotic phenotypic impairment in Clcn5 KO mice. However, hypoxic Clcn5 KO kidneys showed strong fetuin A staining in the PT in addition to luminal signals (Figure 3F, H), providing evidence that the observed cellular fetuin A staining originated purely in the PT.Double immunofluorescence staining of fetuin A and various renal segment markers (Figure 3I-L) confirmed that fetuin A was expressed only in the PT of hypoxic fetal kidneys. Whole-mount in situ hybridization demonstrated fetuin A mRNA synthesis in hypoxic fetal kidneys but not in normoxic kidneys.

[0077] Ahsg contains a putative HIF-binding site that overlaps with the enhancer region. Since fetuin A has been shown to be produced locally in hypoxic fetal kidneys, we evaluated whether Ahsg expression is directly activated by hypoxia. To identify potential HIF-binding sites (hypoxia-response elements - HREs) at the human AHSG locus, we used HIF-1α (HIF-1-alpha) and HIF-2α (HIF-2-alpha) ChIP-seq datasets derived from hypoxic MCF7 cells. A cluster of potential HREs was identified near exon 4 of human AHSG, overlapping with H3K27Ac and H3K4Me1 (chromatin marks of active enhancer elements) and DNase1 hypersensitivity. Another putative HRE was located in the first intron. Screening of 15 Ahsg genes for the presence of consensus HIF binding sites (RCGTG) located 10kb upstream and downstream of ATG revealed peaks 1–5kb downstream of ATG, with an average of 2 HREs per 1kb window. Notably, not only annotated human ChIP-seq HIF sites but also four potential mouse HREs were localized to these peaks. The latter alignment, bearing enhancer marks, revealed a close association with H3K27Ac, ​​H3K4Me1, and DNase1 hypersensitivity.

[0078] Hypoxia activates fetuin A transcription in vitro. Five putative HREs (HREs) of mouse Ahsg and their surrounding DNA, along with nonsense mutations at these sites, were cloned into a luciferase reporter plasmid (Figure 4A). Normal rat renal epithelial (NRK) cells transfected with a reporter plasmid containing only the putative -2kb HRE did not show increased luciferase activity under hypoxic conditions (Figure 4B). Conversely, NRK cells with a reporter plasmid containing the downstream HRE showed a significant increase in luminescence under hypoxia. Mutation of these HREs did not result in an increase in luciferase activity. Furthermore, combining upstream and downstream HREs did not enhance luciferase activity. In summary, HREs located downstream of ATG conferred hypoxia-inducible ability to the mouse Ahsg gene. Finally, fetuin A protein was detected in primary mouse PT cells (pPTCs), NRK cells, and human HK2 cells when cultured under hypoxic conditions (Figures 4C-E). Taken together, these findings identify fetuin A as a novel evolutionarily conserved HIF-dependent target gene.

[0079] Fetuin A deficiency exacerbates CKD progression in hypoxic IUGR kidneys. To investigate how fetuin A induction in fetal hypoxic IUGR kidneys has long-term effects on renal function, urinary protein levels were measured and glomerular filtration rate (GFR) was determined in adult mice (Figure 5A, B). In 9-week-old mice exposed to fetal hypoxia compared to normoxic mice, GFR decreased and proteinuria increased. Furthermore, evaluation of fibrotic tissue remodeling revealed that in the kidneys of 9 fetal hypoxic Ahsg KO mice, compared to hypoxic wt and normoxic controls, more collagen structures extended deeper into the subcortical region, and the expression levels of fibrosis markers were highest (Figure 5C-G). Importantly, these results clearly demonstrate that renal function was systematically susceptible in Ahsg KO mice, showing an additive effect of hypoxia and fetuin A deficiency.

[0080] Fetuin A deficiency promotes the accumulation of calcium mineral particles and macrophages in hypoxic IUGR kidneys. Adult fetuin A knockout mice are prone to soft tissue calcification, but no overt calcification was detected in the kidneys of hypoxic Ahsg knockout mice, as assessed by von Kossa staining. To further investigate whether fetuin A expression in fetal hypoxic kidneys affects mineralized matrix processing, the presence of calcium-containing nanoparticles was demonstrated by incubating freshly cut kidney sections from E18.5 embryos with ATTO488 fluorescently labeled fetuin A (488-FA). Due to the high affinity binding of fetuin A to calcium phosphate, 488-FA staining is more sensitive to the detection of calcium-containing matrix and cellular remnants than commonly used mineral staining protocols. Therefore, positive 488-FA staining in the absence of von Kossa or alizarin red staining also highlights simply calcium-enhanced structures, including amorphous calcium phosphate aggregates, often preceding overt calcification. 488-FA staining revealed strong labeling of PT, the site of major calcium reabsorption and therefore similar mineralization matrix processing, in normoxic wt kidneys (Figure 6A). The intensity of PT staining decreased in hypoxic wt kidneys and increased in hypoxic Ahsg KO kidneys, suggesting increased and decreased mineralization matrix turnover, respectively (Figures 6B, C). In addition, only hypoxic Ahsg KO kidneys showed a granular staining pattern in the papillary region and, to a lesser extent, in the renal region of the outer cortex (arrowheads in Figures 6D-I), indicating massive mineral deposition in the absence of endogenous fetuin A. Excessive amounts of mineral or cellular debris are often found in sites of increased cell death. Indeed, TUNEL staining confirmed apoptosis in hypoxic Ahsg KO kidneys but not in normoxic or hypoxic wt kidneys. Severed caspase-3 immunohistochemistry in hypoxic Ahsg KO kidneys further demonstrated cell death in these kidneys. Finally, the accumulation of mineralized material triggered an inflammatory response, leading to macrophage infiltration, tissue damage, and fibrosis.Staining for the macrophage marker F4 / 80 revealed a significant accumulation of these cells only in hypoxic Ahsg knockout compared to wt kidney, suggesting the protective effect of fetuin A (Figure 6J-N). Taken together, these data provide the first demonstration of the role of fetuin A in the binding and clearance of the mineralized matrix in the kidney, protecting tissue integrity.

[0081] Fetuin A attenuates the hypoxia-induced expression of fibrosis markers by antagonistizing TGF-β signaling. Despite similar mRNA levels of transforming growth factor beta-1 (Tgfb1), a potent fibrosis inducer, the kidneys of fetal hypoxia-Ahsg KO mice generally showed higher levels of fibrosis marker expression compared to fetal hypoxia-wt animals (Figure 5). 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 increased even further under hypoxia-induced culture conditions (Figures 7A-D, G). This hypoxia-induced increase was blunted by supplementation of the culture medium with fetuin A (Figures 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 compared to wt cells (Figure 7H, I), indicating that Ahsg KO cells responded more actively to TGF-β1. In contrast, the addition of fetuin A before TGF-β1 treatment reduced Smad3 phosphorylation. These findings are consistent with previous reports describing fetuin A as a TGF-β2 receptor mimetic and cytokine antagonist. Taken together, the results suggest that fetuin A reduces hypoxia-11-induced renal fibrosis by directly antagonizing TGF-β1 signaling, in addition to its protective role as a mineral chaperone.

[0082] Fetuin A significantly reduces the expression of fibrosis markers in vivo in the kidney after ischemia-reperfusion injury. Next, we experimentally explored the therapeutic potential of fetuin A supplementation in the treatment of kidney injury. For this purpose, we used a well-established mouse model of unilateral ischemia-reperfusion injury. This IRI model, as its name suggests, best mimics ischemia-reperfusion lesions in the kidney after cardiovascular surgery, kidney transplantation, or removal of a renal tumor. It is simple and reproducible. Ischemia was induced in the left kidney by clamping the renal vessels for 30 minutes, with the right kidney used as a control. The accumulation of calcium mineral particles and macrophages in the hypoxic IUGR kidney demonstrated for the first time that ischemia-reperfusion actually induces calcium mineral nanoparticle deposition in the postoperative kidney but not in the contralateral control kidney (Figure 8). In addition, treatment of IRI mice with fetuin A compared to 0.9% NaCl showed that by day 5, fetuin A treatment significantly reduced specific fibrosis markers (collagen Col1a1, Col3a1, and Col6a1) in ischemic, injured kidneys compared to animals treated with 0.9% NaCl (Figure 9A, B, C). In contrast, fetuin A supplementation had no effect on the expression of non-collagen fibrosis markers Acta2, Fn1, or Vim (Figure 9D, E, F). Therefore, fetuin A supplementation attenuates the expression of fibrosis markers after ischemia-reperfusion injury. In summary, these in vivo experiments clearly establish the role of fetuin A as a mineral chaperone protecting the kidney from hypoxia-responsive IR injury.

[0083] Prophylactic administration of human fetuin A in mice undergoing ischemia-reperfusion injury (IRI) reduced tissue damage and decreased fibrotic remodeling. Fetuin A 900 μg was intravenously injected 24 hours and 3 hours prior to unilateral IRI in the left kidney (20-minute ischemia time). PBS was injected into control animals. Kidneys were harvested 24 hours after surgery, with the right kidney used as control. Expression levels of early injury markers Kim1 (Figure 10A), Krt18 (Figure 10B), Krt20 (Figure 10C), and Lcn2 (Figure 10D) were shown to be reduced in IRI kidneys pre-treated with fetuin A. This already indicates that pre-treatment with fetuin A mitigates tissue damage from reperfusion injury (IRI). In addition, the decrease observed in Vim expression (Figure 10G) indicated a reduction in epithelial-mesenchymal transition (EMT). Collagen remodeling is a relatively late event during fibrous remodeling, and it did not play a significant role 24 hours after IRI, resulting in no unexpected changes in Col1a1 (Figure 10H), Col3a1 (Figure 10I), and Col6a1 (Figure 10J). Arg1 (Figure 10K) expression was suppressed in fetuin A-pretreated mice, indicating a reduced need for collagen synthesis. Arg1 is important for increased collagen synthesis because it is required for the supply of L-proline, the main component of collagen. Furthermore, pretreatment with fetuin A reduced the expression of chemokines Ccl2 (Figure 10L), Il-6 (Figure 10M), and Tnf-α (Figure 10N), which are involved in damage signaling and repair. In conclusion, these experiments, particularly the early injury markers, demonstrate that prophylactic administration of human fetuin A in mice suffering ischemia-reperfusion injury (IRI) suppressed injury and reduced fibrotic remodeling, even at relatively low concentrations of fetuin A.

[0084] The presence of endogenous fetuin A in mouse kidney tissue after intravenous (iv) administration. Human fetuin A was not detected in PBS-injected samples (Figure 11A, lanes G-L), confirming the specificity of the sc-133146-HRP anti-fetuin A antibody to human fetuin A rather than mouse fetuin A. PBS-injected IRI kidneys (Figure 11A, lanes J-L) showed the highest levels of mouse fetuin A among all samples, indicating a higher degree of biomineralization and tissue damage in these kidneys. Kidney samples from mice injected with human fetuin A protein showed a strong band of human fetuin A (Figure 11A, lanes A-F), confirming the presence of fetuin A in kidney tissue after intravenous administration. Endogenous mouse fetuin A was elevated only in PBS-injected IRI kidneys compared to all other samples, indicating a higher degree of biomineralization and tissue damage in these kidneys (Figure 11B). Human fetuin A was not detected in PBS-injected kidney samples, confirming the specificity of the DFTA00 ELISA kit from the R&D system (bio-techne) to human fetuin A rather than mouse fetuin A (Figure 11C). Fetuin A-injected IRI kidneys showed higher tissue levels of human fetuin A and a greater degree of biomineralization in ischemic kidneys compared to unsurgery control kidneys (Figure 11C). Endogenous fetuin A was present in the proximal tubules of mice after intravenous administration (Figures 11D-F). While not bound by any particular theory, it could be hypothesized that fetuin A can be taken up from the blood to the site of tissue damage, antagonizing further tissue destruction and reducing its concentration in the blood. In conclusion, these experiments demonstrate that intravenous administration of fetuin A results in an increase of fetuin A in kidney tissue.

[0085] Time course of human fetuin A in mouse serum after intravenous administration. After 1 day, the detectable amount of human fetuin A in serum decreased compared to 1 minute or 1 hour after injection (Figure 12A), and after 1 week, human fetuin A was undetectable. Mouse fetuin A was not detected with the sc-133146-HRP anti-human fetuin A antibody, as can be inferred from the fact that the PBS-injected animals did not signal. Normalized ELISA data using a human fetuin A-specific DFTA00 ELISA kit showed a decrease in intravenously injected human fetuin A in mouse serum samples (Figure 12B, C). Compared to the time of injection (1 minute), the amount of circulating human fetuin A decreased to 80% after 1 hour and to 8.5% after 1 day. After 1 week, human fetuin A was no longer detectable, as in PBS-injected control mice (black circles). In conclusion, these experiments indicate that injections should be repeated daily to maintain the presence of circulating human fetuin A.

[0086] method Animals. Breeding, genotyping, and all animal experiments are conducted in accordance with Swiss federal law on animal welfare and approved by local authorities (Canton of Bern BE96 / 11, BE105 / 14, and BE105 / 17). Ahsg tm1Mbl Mouse and Clcn5 tm1Gug All mice, including the one mentioned, were maintained on a C57BL / 6 background, with ad libitum access to food and water, and housed in IVC cages with a 12-hour light-dark cycle.

[0087] Induction of hypoxia in pregnant mice. For timed mating, the vaginal plug of breeding females was checked every morning, and if present, this point was set as gestation day (E) 0.5. Ahsg KO mice were obtained from heterozygous breeding pairs, and heterozygous and wt littermates were also produced and used as controls. Daily food intake (weight difference between the food initially offered and the food remaining after 24 hours) and maternal weight gain were recorded during pregnancy from E0.5 to E18.5. To induce hypoxia, E13.5 pregnant mice were moved to a hypoxic glove box (Coy Laboratory Products, Grass Lake, USA). The following day, the oxygen content was gradually reduced to 10% over 6-8 hours, intermittently stopping at 16% and 12.5%, to allow the animals to adapt to increasing hypoxic conditions. Adequate air circulation was maintained with a room fan. CO2 levels were kept low by chelating excess CO2 with a cartridge filled with soda lime (Sigma, 72073) connected to an air circulation system. Excess moisture was absorbed with granular silica gel orange (Sigma, 1.01969) and replaced daily. Mice in the calorie-restricted group were fed a diet equivalent to the average food intake of mice in the hypoxic group. Hypoxia-pregnancy mice or control mice were euthanized at E18.5, and the fetuses and placentas were collected, weighed, and prepared for further analysis. Fetal kidneys were dissected in PBS using a Leica M80 stereoscope. Kidneys from 3-4 week old normoxic mice were isolated for the preparation of primary proximal tubular cells (pPTCs). Renal function (GFR and proteinuria) was evaluated in 9-week-old fetal hypoxia-pregnancy

[0088] Induction and administration of ischemia for therapeutic treatment experiments. Mice were anesthetized with isoflurane for the experiments shown in Figures 8 and 9. A midline abdominal incision was made in the deeply anesthetized animals. Ischemia was induced in the left kidney by clamping the renal vessels for 30 minutes, and the right kidney was used as a control (Figure 9). Immediately after surgery, mice received initial treatment by intraperitoneal injection of either physiological NaCl solution or bovine fetuin A monomer (100 μg / g body weight). Injections were repeated daily for 4 days. Kidneys were isolated and analyzed on day 5. NaCl solution: sodium chloride ≪Bichsel≫ 0.9% (154 mM); (REF100 0 090, Bichsel, Interlaken, CH). Fetuin A solution: 0.338 EU / ml LPS, 10 mM Na3PO4, 8 g / l NaCl (137 mM).

[0089] Induction and administration of ischemia for prophylactic (prevention) experiments. For the experiment in Figure 10, mice were intravenously injected with 900 μg of fetuin A 24 hours and 3 hours before unilateral ischemia. PBS was intravenously injected into control animals. Mice were anesthetized with isoflurane to induce ischemia. In deeply anesthetized animals, a midline abdominal incision was made, and ischemia was induced in the left kidney by clamping the renal vessels for 20 minutes. The right kidney was used as an unsurgery control. The kidneys were harvested 24 hours after surgery. The average body weight of the mice in the experiment 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.

[0090] Transcutaneous evaluation of glomerular filtration rate (GFR). Glomerular filtration rate (GFR) was determined in conscious animals as described by Schreiber, A. et al., Transcutaneous measurement of renal function in conscious mice. Am J Physiol Renal Physiol 303, F783~788 (2012). In short, the plasma clearance of FITC-sinistrin (Fresenius-Kabi, LI9830076) was measured across the skin using a light-emitting diode with a maximum emission of FITC at 470 nm and a photodiode that detects fluorescence with maximum sensitivity at 525 nm. The decrease in fluorescence intensity over time was then converted to GFR.

[0091] Proteinuria. Urine protein content was determined using the Bradford assay. 3 μl of urine and 150 μl of 1× Bradford reagent were mixed and incubated at RT for 5 minutes, and the absorbance was measured at 595 nm (5× 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, and then adjusting the total volume to 100 ml with ultrapure water). Urine creatinine content was determined using the Jaffe method. 10 μl of 1:10 diluted urine was mixed with 100 μl of creatinine reagent, incubated at RT for 10 minutes, and the absorbance was measured at 510 nm (creatinine reagent consisting of 10 mM picric acid and 250 mM NaOH, pH 13). Finally, the protein-creatinine ratio was calculated for each sample.

[0092] Glomerular count. 100 μm Z-stacked images of whole-mount E18.5 kidneys stained with nephrine (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 Downsample LoG detector was set to an estimated blob diameter of 80.0 pixels with a pixel threshold of 16 and a downsampling factor of 2. The number of spots per frame was added to the mouse to calculate the number of glomeruli per kidney. A distance of 100 μm between frames was selected to avoid double counting of the same glomerulus in consecutive images, resulting in an average glomerular diameter of 80 μm.

[0093] Microarray analysis. Total RNA was isolated from E18.5 kidneys of male hypoxia, normoxic, and calorie-restricted mice 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. 100 ng of total RNA sample was treated with the Ambion® WT Expression Kit (4411973, Life Technologies). 5.5 μg of cDNA was fragmented and labeled with the GeneChip® WT End Labeling Kit (901525, Affymetrix). 2.3 μg of biotinylated fragments were hybridized to an Affymetrix Mouse Gene 1.0 ST array at 45°C for 16 hours, washed, and stained according to the protocol described in the Affymetrix GeneChip® Expression Analysis Manual (Fluid Protocol FS450_0007). The array was scanned with an Affymetrix GeneChip® Scanner 3000 7G, and the raw data was extracted from the scanned images and analyzed using the Affymetrix Power Tools software package. The quality of hybridization was evaluated using the Affymetrix Expression Console software (version 1.1.2800.28061). Normalized expression signals were calculated from Affymetrix CEL files using the Robust Multi-Array Means Algorithm (RMA) algorithm. Differentially hybridized features were identified using the R Bioconductor package "limma," which implements a linear model of microarray data. P-values ​​were corrected for multiple trials using the Bengiamini-Hochberg method to control the false detection rate (FDR). Probe settings showing at least a 1.3-fold change and an FDR < 0.05 were considered significant. Differential E18.5 expression levels between hypoxic, normoxic, and calorie-restricted kidneys were mapped using Heatmapper (http: / / www.heatmapper.ca) with mean linkage and Euclidean distance measurements.For feature annotation, GO terminology analysis was performed using the DAVID platform (https: / / david.ncifcrf.gov).

[0094] RT-qPCR. Total RNA was isolated using TRIzol® reagent (Invitrogen 15596026) according to the manufacturer's protocol. RNA concentration and quality were determined using a Nanodrop 1000 spectrophotometer (ThermoFisher Scientific, Switzerland), and 1000 ng was transcribed to cDNA using the 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 either a TaqMan gene expression assay (ThermoFisher) or a FAM-labeled UPL probe (Roche) along with corresponding gene-specific primers and a TaqMan Fast Universal PCR Master Mix (Applied Biosystems, 4352042). Data analysis was performed using Microsoft Excel. 2 (-ΔCt) The relative expression levels of RT-qPCR were calculated using the specified method.

[0095] Identification of HIF-binding sites. To identify putative HIF-binding sites, 20kb genomic regions (10kb upstream and downstream of the start codon) overlapping with the Ahsg locus in 15 different species (cat, chicken, chimpanzee, cattle, dog, chimaera, horse, human, mouse, pig, rabbit, rat, sheep, xenopus, and zebrafish) were analyzed in the JASPAR database (http: / / jaspar.genereg.net). The relative profile score threshold was set to 90%. For enrichment analysis, putative sites for all species were clustered into 1kb windows. HREs with relative scores >0.9, >0.93, and >0.97 were shown. For the alignment of human HIF-alpha sites identified by ChIP-seq of hypoxic MCF7 cells possessing the activity regulation mark of the AHSG locus (see Schodel J, Oikonomopoulos S, Ragoussis J, Pugh CW et al., Blood 2011 / June 9;117(23):e207~17;Series GSE28352;https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE28352), the HIF-1-alpha and HIF-2-alpha datasets (including the AHSG locus) containing chr3:180,000,000-190,000,000 were converted to 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). To this alignment, we added the following datasets: layered chromatin marks frequently found near regulatory elements in seven cell lines (H3K27Ac and H3K4Me1, ENCODE) and open chromatin from hypoxic MCF7 cells (DNaseI HS, ENCODE).For the mouse Ahsg locus, potential HIF binding sites identified by JASPAR were aligned with datasets derived from the liver, heart, and kidney of 8-week-old mice (DNaseI HS, ENCODE / UW; H3K27Ac and H3K4Me1, ENCODE / LICR), and the mean signal intensity was shown (bar graph, autoscale, log transformation, smoothed (16 pixels)).

[0096] Molecular cloning. For luciferase assays, the upstream portion of a 2.5 kb promoter fragment of mouse Ahsg ATG was amplified from genomic DNA (C57BL / 6) using specific primers and PrimeSTAR® GXL DNA polymerase (Takara, R050A). 500 bp promoter fragments (wt and mutant) and 500 bp intron sequence fragments (wt and mutant) were synthesized by IDTDNA (https: / / eu.idtdna.com / pages). The promoter fragments were inserted into the pGL3-basic-P2P-607 plasmid using NcoI and SacI restriction enzymes (both NEB, R3193S and R3156S). The intron fragments were inserted into pGL3-basic vectors or pGL3-basic vectors containing the promoter fragments using BamHI and Sall restriction enzymes (both NEB, R3136S and R3138S). For in-situ hybridization, cDNA of exons 2-5 of mouse Ahsg was obtained from IDTDNA and cloned into pBluescriptll KS- using Spel and EcoRI restriction enzymes (both NEB, R3133S, and R3101S).

[0097] Luciferase assay. 24 hours after transfection, cells were washed twice with PBS, lysed on ice for 20 minutes (250 mM KCl, 50 mM Tris / H3PO4 pH 7.8, 10% glycerin, 0.1% NP40), and centrifuged at 14000 rpm at 4°C for 10 minutes. 10 μl of the supernatant was used for each reaction. Reaction solutions (luciferase: 100 μl consisting of 25 mM Tris / H3PO4 pH 7.8, 10 mM MgSO4, 2 mM ATP pH 7.5, 50 μM luciferin; sea cucumber: 100 μl consisting of 50 mM Tris / HCl pH 7.6, 100 mM NaCl, 1 mM EDTA, 0.5 μM coelenterazine) were injected and their activity measured using a Fluoroskan Ascent FL (ThermoFisher). Each sample was measured in a double-dash configuration, and luciferase activity was normalized relative to the activity of *Cortinarius luteus*.

[0098] Whole-mount in situ hybridization. E18.5 kidneys were fixed in 4% PFA, dehydrated, and stored in methanol at -20°C. In situ hybridization using digoxigenin-labeled riboprobes was performed 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 mix (Roche, 11175025910) and T3 or T7 RNA polymerase (both Roche, 11031163001 or 10881767001). The DIG-labeled probes (Roche, 11093274910) were detected using alkaline phosphatase conjugate antibodies.

[0099] TUNEL staining. Fragmented DNA from apoptotic cells was detected using the Promega DeadEnd Colorimetric TUNEL System (G7360) according to the manufacturer's instructions.

[0100] Histochemistry. For immunohistochemistry, PFA-fixed paraffin-embedded tissue sections were rehydrated, and endogenous peroxidase was blocked by incubating the slides in 1.5% H2O2 solution (0.02 M citrate, 0.06 M Na2HPO4) in the dark at RT for 15 minutes. Antigen retrieval was performed by boiling in Tris-EDTA buffer pH 9 for 20 minutes, then slowly cooling to RT. After blocking in 2% BSA in PBS at RT for 1 hour, the sections were incubated o / n at 4°C with primary antibody in blocking solution. After three washes in PBS, the sections were incubated at RT for 1 hour with HRP-conjugated secondary antibody (mouse or rabbit: Dako EnVision+ System (K4001 or K4003) from Agilent; goat: SCBT, sc-2304). After three washes in PBS, the signal was generated using DAB (Agilent, K3468). Sections were counterstained with Harris hematoxylin solution (Sigma, HHS16), dehydrated, and mounted in Eukitt medium (Sigma, 03989). For picrosilius red staining of collagen, defatted and rehydrated tissue sections were incubated at RT for 1 hour in staining solution (0.5 g of Direct Red 80 in saturated picric acid solution (both Sigma, P6744 and 365548)). After two washes in acidified water (0.5% glacial acetic acid), the sections were dehydrated and mounted in Eukitt medium.

[0101] Immunofluorescence staining. Frozen sections were fixed in 4% PFA at RT for 10 minutes, washed twice in PBS, and permeabilized by incubation at RT for 10 minutes in PBST (0.1% Triton X-100 in PBS). After blocking at RT for 1 hour in PBS with 10% FCS and 0.5% Tween-20, the sections were incubated o / n at 4°C with the primary antibody in the blocking solution. After three washes in PBS, the sections were incubated at RT in the dark for 1 hour with the fluorescent conjugate secondary antibody in the blocking solution. DNA was stained with DAPI 1:5000 in PBS. Sections were mounted in MOWIOL solution (2.4 g of MOWIOL 4-88 reagent (Merck, 475904) in 6 g of glycerin and 18 ml of 0.13 M Tris pH 8.5). For whole-mount immunofluorescence staining of E18.5 kidneys, the iDisco staining protocol (https: / / idisco.info / idisco-protocol / ), including methanol pretreatment, was applied. An incubation time of n=1 day and a solution volume of 1.6 ml were used for the relevant steps. Kidneys were mounted on 8-well glass chamber slides (ThermoFisher, 154534) and imaged immediately.

[0102] Detection of calcium fluorescence. Thick frozen sections (30 μm) were incubated in the dark at RT for 60 minutes with ATTO 488 fluorescently labeled fetuin A 10 ng / ml (in calcium-free PBS), rinsed three times with PBS, and mounted in MOWIOL solution. Nuclei were counterstained with DAPI.

[0103] Imaging. Fluorescence imaging was performed using an IMIC digital microscope (FEI, model 4001) with a Polychrome V light source, and Orca-R from Hamamatsu. 2The images were taken using a camera controller (C10600) and live acquisition software (FEI, version 2.6.0.14). Image analysis was performed using offline analysis software (FEI). Brightfield imaging was performed using a Nikon E600 microscope equipped with Nikon objective lenses (Plan Fluor ELWD 20× / 0.45, Plan Apo 40× / 1.0 Oil, and 60× / 1.40 Oil) with a digital site DS-UE camera controller and DSRi1 camera (both Nikon). Image analysis was performed using Nikon software NIS Elements 4.0.

[0104] ELISA. For example, mouse fetuin A levels in serum or kidney tissue were determined using the mouse-specific fetuin A / AHSG Quantikine ELISA kit (R&D, MFTA00) according to the manufacturer bio-techne. For example, human fetuin A levels in serum or kidney tissue were determined using the human-specific fetuin A / AHSG Quantikine ELISA kit (R&D, DFTA00) according to the manufacturer bio-techne.

[0105] 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, NRK cells were transfected with a luciferase reporter plasmid and pCMV-Sea Mushroom (10% of total transfected DNA, used for normalization) using jetPrime® reagent (Polyplus, 114-07). Six hours after transfection, cells were stimulated with 1 mM DMOG (Echelon Biosciences, F-0010), and collected 24 hours after transfection. For hypoxia, cell cultures were maintained in 0.2% oxygen for 48 hours. Primary proximal tubular cells (pPTCs) were isolated from the kidneys of 3-4 week old mice (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)). Simply put, proximal tubular fragments were obtained by digesting renal cortical tissue with collagenase and filtering it 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, and 1% NEAA, and in renal epithelial cell growth medium (REGM) supplement (Lonza, CC-4127). Serum from Ahsg KO mice was used instead of FBS. Depending on the density, pPTCs were divided and re-seeded once using Accutase solution (Sigma, A6964). For hypoxia, cell culture was performed in 0.2% oxygen for 48 hours. Fetuin A (Sigma, F3385) or bovine serum albumin (BSA, Sigma, A3059) 100 μg / ml was added to the culture medium 48 hours before the end of the experiment. Cells were starved for 24 hours before treatment with rmTGF-β1 (R&D, 7666-MB) 5 ng / ml for 5 minutes.

[0106] Western blot analysis was performed. Total protein solubilization was obtained using RIPA buffer (Sigma, R0278) supplemented with a protease inhibitor (Roche, 11836153001). Proteins were separated by SDS-PAGE and blotted onto a PVDF membrane (ThermoFisher, 88518). After blocking with 5% milk in TBST, the membrane was incubated with primary antibody at 4°C o / n and then incubated with HRP-conjugated secondary antibody at RT for 1 hour. Signals were detected using ECL (GE Healthcare, RPN2106) or SuperSignal (ThermoFisher, 34076) depending on signal intensity. Concentration analysis was performed using the open-source image processing software Fiji (ImageJ, version 2.0.0-rc69 / 1.52i, https: / / imagej.net / Fiji).

[0107] Calculation of the concentration of bovine fetuin A administered intraperitoneally in mice. The reference blood volume of 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 the calculation. The calculated molecular weight of bovine fetuin A (UniProtKB-P12763(FETUA_BOVIN)) is 38.4 kDa. Therefore, if all of the injected fetuin A is present in the blood, or if fetuin A is administered intravenously, the blood concentration will reach 38 μm. Furthermore, fetuin A undergoes post-translational modification. Here, glycosylation plays a central role, in particular, and the molecular weight increases to 51-67 kDa. Therefore, a molecular weight of 60 kDa was used for the calculation (1 Da = 1 g / mol). If all of the injected fetuin A is present in the blood, or if fetuin A is administered intravenously and the injection volume is based on 100 μg / g body weight, the concentration in the blood of the injected mouse will be 24.3 μM:

number

number

[0108] Calculation of the concentration of intravenously administered human fetuin A in mice. The reference blood volume of 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 the calculation. The calculated molecular weight of human fetuin A (UniProtKB-P12763(FETUA_BOVIN)) is 38.4 kDa. Furthermore, fetuin A undergoes post-translational modifications. Here, glycosylation plays a central role, particularly increasing the molecular weight to 51-67 kDa. Therefore, a molecular weight of 60 kDa was used for the calculation. In the prophylactic treatment experiment, the average body weight of the mice was 22 g. For a mouse weighing 22 g, the corresponding blood volume was approximately:

number

[0109] This is an injection of human fetuin A 900 μg, which is,

number

[0110] Calculation of human fetuin A concentration and dosage in humans. The normal blood volume for adult humans is 77 ml / kg (male) and 65 ml / kg (female), with an average volume of 71 ml / kg. The molecular weight of human fetuin A (UniProtKB-P02765 (FETUA_HUMAN)) is generally equivalent to that of bovine fetuin A, i.e., approximately 60 kDa (due to glycosylation). For administration of fetuin A at 10 mg / kg (iv), 25 mg / kg (iv), and 50 mg / kg (iv) to adult humans, the resulting human blood concentrations are:

number

[0111] Similarly, the dose corresponding to the above concentration of 9.7 μm can be calculated:

number

[0112] Similarly, the dose corresponding to the above concentration of 9.9 μm can be calculated:

number

[0113] Data analysis. Statistical analyses and graphs were performed using Prism 7 (https: / / www.graphpad.com). Two groups were compared by t-test, and multiple groups were compared by one-way ANOVA (Tukey's 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.

Claims

1. A pharmaceutical composition for use in the treatment of renal impairment, comprising fetuin A and optionally at least one pharmaceutically acceptable carrier, The pharmaceutical composition wherein the renal impairment is selected from the group consisting of ischemic renal injury, hypoxia-related injury of the kidney, renal ischemia-reperfusion injury, delayed graft function, organ rejection of a kidney graft, kidney tissue damage resulting from kidney transplantation, inflammation resulting from kidney transplantation, and combinations thereof.

2. A pharmaceutical composition for use in the treatment of renal impairment according to claim 1, wherein the renal impairment is caused by hypoxia during surgery.

3. The pharmaceutical composition according to any one of claims 1 to 2, wherein fetuin A is human fetuin A.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein fetuin A is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO:

1.

5. Fetuin A is a pharmaceutical composition according to any one of claims 1 to 4, comprising or derived from Sequence ID No.

1.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein fetuin A is recombinantly expressed to provide post-translational modifications.

7. The pharmaceutical composition is administered to a subject by injection or inhalation, as described in any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is administered by intravenous injection to a target.

9. Fetuin A is administered to the subject at a concentration of 1 to 200 mg / kg (body weight), 5 to 100 mg / kg (body weight), or 10 to 50 mg / kg (body weight), as per any of claims 1 to 8. The pharmaceutical composition described in item 1.

10. The treatment comprises adjusting the level of calcification in kidney tissue, the pharmaceutical composition according to any one of claims 1 to 9.

11. A pharmaceutical composition according to any one of claims 1 to 10, wherein the treatment comprises the removal of calcium mineral deposits in kidney tissue.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein at least two or at least three doses of fetuin A are administered.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein at least one dose of fetuin A is administered before, during, or after an ischemic accident and / or surgery.

14. The pharmaceutical composition according to claim 13, wherein at least one dose of fetuin A is administered within 48 hours prior to an ischemic event and / or surgery, or at least one dose of fetuin A is administered within 48 hours after an ischemic event and / or surgery.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein fetuin A is administered in divided doses over a total duration of 1 to 50 days.

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