WO2004009102A1

WO2004009102A1 - Use of densin-like compounds

Info

Publication number: WO2004009102A1
Application number: PCT/FI2003/000579
Authority: WO
Inventors: Harry Holthöfer; Heikki Ahola
Original assignee: Glomega Inc. Oy
Priority date: 2002-07-24
Filing date: 2003-07-23
Publication date: 2004-01-29
Also published as: AU2003246752A1; FI20021406A0

Abstract

The invention is related to the use of densin-like compounds expressed in glomerular podocytes as well as nucleic acids encoding densin and binding substances specifically recognizing and binding to or interacting with densin-like compounds which are useful for manufacturing products including test kits for diagnosing and treating glomerular diseases manifesting with proteinuria due to hereditary, infectious, inflammatory, toxic or metabolic reasons, IgA nephropathy, membranous, diabetic, crescentic and minimal change nephropathies as well as nephropathy associated with hypertension and infections including HIV associated nephropathy.

Description

USE OF DENSIN-LIKE COMPOUNDS

The Technical Field of the Invention

The present invention relates to the use of densin-like compounds or fragments thereof as well as nucleic acids encoding said densin-like compounds or fragments present in the kidney for manufacturing products useful for diagnosing and treating glomerular diseases manifesting with proteinuria. It is also related to an in vitro method and a test kit for diagnosing the presence, predisposition or risk of developing a glomerular disease as well as a progress of healing.

Background of the Invention

Densin belongs to the LAP [leucine-rich repeats (LRRs) and PDZ domains] protein family with leucine-rich repeats, a sialomucin domain and PDZ domain. (Apperson et al., J Neurosci 16:6839-52, 1996). LAPs are found in distinct epithelia and postulated to be involved in the maintenance of the cell shape and the apical-basal polarity. Densin localizes mainly at the site of cellular synapses as in the dendrites in hippocampal neuronal cultures.

The ability of glomerular epithelial cells, podocytes, to form foot processes with unique intercellular structures called slit diaphragm reflects a special role in glomerular filtration. The composition of the protein complex forming the slit diaphragm has been poorly known. However, the demonstration of NPHS1 (Kestila et al., Molecular Cell 1:575-582, 1998), the gene encoding nephrin, and its expression in the slit diaphragm (Holthofer et al., Am J Pathol 155:1681-7, 1999) have elucidated the molecular structure of the slit diaphragm.

Glomerular diseases are known to manifest with proteinuria in the end stages of the disease. However, the pathophysiology and the glomerular permeability changes in glomerular diseases remain poorly understood and little progress has so far been achieved in the targeted treatment of the diseases. Thus, there is a great need of methods and test kits for accurate diagnosis of glomerular diseases as well as methods and means for prophylactic, preventive and therapeutic treatment thereof, hi addition, the same applies for other primary or secondary diseases of the kidney presenting with proteinuria.

New methods and means for studying and diagnosing glomerular diseases are needed. The present methods for diagnosing glomerular diseases are not precise enough. There is a need for new diagnosing methods and test kits for accurate diagnosis.

The present invention provides a solution to the above defined problems by offering a new approach including methods and means for diagnosing and treating glomerular diseases based on the use of densin-like compounds, nucleic acids encoding the same as well as binding substances specifically recognizing and binding to or interacting with the same.

Thus, the objective of the present invention is to provide new methods and means for treating or diagnosing, evaluating the efficacy of treatment, screening large populations for the susceptibility of glomerular diseases or disorders manifesting with proteinuria.

The Summary of the Invention

The present invention relates to the use of densin, including densin-like proteins, nucleotide sequences encoding such and binding substances specially recognizing and binding to or interacting with such. They are applicable for manufacturing products useful for diagnosing and treating glomerular diseases manifesting with proteinuria. Particularly an in vitro method and a test kit for diagnosing the presence, predisposition or risk of developing a glomerular disorder as well as progress for healing is disclosed.

The characteristic features of the present invention are defined in detail in the claims.

A Short Description of the Drawings

Figure 1 depicts the expression of densin mRNA in the rat brain, in the cultured human podocytes (pod.), in the normal human kidney cortex and in the normal human glomerulus (glom.). RT-PCR results were verified by direct sequencing of the PCR products. The amplification remains negative in the control samples. For controlling the quality of RT reactions PCR with β-actin specific primers was performed.

Figure 2 A depicts immunoblotting of human brain (1), cultured podocytes (2) and glomerular lysates (3) with an affinity purified antibody to the extracellular domain of densin. A band of 185 kDa can be seen in the brain (1) and the podocytes (2) whereas a 210 kDa band is detected in the glomeruli (3). Molecular mass markers appear on the left.

Figure 2B depicts human glomerular lysate immunoprecipitated with antinephrin antibody #1109 showing coprecipitation of densin. Such precipitation with the control sample, Protein A sepharose 4B, cannot be seen.

Figure 3 A depicts a frozen section of the normal human kidney stained with affinity purified densin antibodies and shows reactivity in the glomerulus and faintly also in the proximal tubules.

Figure 3B depicts a higher magnification of a frozen section of the normal human kidney stained with affinity purified densin antibodies and shows reactivity in the glomerulus and faintly also in the proximal tubules; a finely granular linear staining can be seen.

Figure 4 depicts immunoblotting of human urine samples from type I diabetic patients with the affinity purified densin antibody. Lanes (1) and (2) contain samples from patients with severe albumin excretion (patients with prolonged glomerular disease); lanes (3) and (4) contain samples from patients with normal albumin excretion (patients without glomerular damage).

Figure 5 shows AF 125521, a rat nephrin sequence (SEQ ID NO:l).

Figure 6 shows AF 035835, a human nephrin sequence (SEQ ID NO:2). Figure 7 shows AF 126957, a partial coding sequence of human nephrin sequence lacking the exon 24 (SEQ ID NO:3).

Figure 8 shows AF 434715, a human densin sequence (SEQ ID NO:4).

Figure 9 shows U66707, a rat densin sequence (SEQ ID NO: 5).

Figure 10A shows immunoelecfronmicroscopy with antidensm antibody showing specific localization at the slit diaphragm domains. The sample was embedded with Lowicryl. Abbreviations: GBM, glomerular basement membrane; US, urinary space.

Figure 10B shows immunoelectronmicroscopy (cryoEM) with antidensin antibody showing specific localization at the slit diaphragm domains.

Figure 10C shows immunoelectronmicroscopy with antidensin antibody showing specific localization at the slit diaphragm domains. The sample was embedded with Lowicryl.

Figure HA shows semiquantitative RT-PCR of normal (NH) and CNF kidney (CNF). Increased densin mRNA expression in CNF cortex is detected.

Figure 11B shows Western blotting of normal (NH) and CNF kidney (CNF). Upregulation of densin protein in CNF is shown by immunoblotting with antidensin antibodies.

The Detailed Description of the Invention

Definitions

The terms used in the present invention have the meaning they have in the fields of medicine and diagnostics, including immunology, immunochemistry, biochemistry, pharmacology and recombinant DNA technology. Some terms in the present invention are, however, used with a somewhat deviating or broader manner. Accordingly, in order to avoid uncertainty caused by terms with unclear meaning some of the terms used in this specification and in the claims are defined in more detail below.

The term "glomerular disease" includes diseases manifesting with proteinuria due to hereditary, infectious, inflammatory, toxic or metabolic reasons including but not restricted to IgA nephropathy, membranous, diabetic, crescentic and minimal change nephropathies as well as nephropathy associated with hypertension and infections including HIV associated nephropathy.

The term "densin" in the present invention means densin proteins but also includes other densin-like compounds. Particularly, densin means densin expressed in the glomerulus or cultured podocytes appearing as a 210 kDa band in immunoblotting studies and fragments or complexes containing repeats thereof.

Densin belongs to the LAP [leucine-rich repeats (LRRs) and PDZ domains] protein family with leucine-rich repeats, a sialomucin domain and PDZ domain. (Apperson et al., J Neurosci 16:6839-52, 1996), LAPs are found in distinct epithelia and postulated to be involved in the maintenance of cell shape and the apical-basal polarity. This is also believed to be the function of densin also in the podocytes, the glomerular epithelial cells. The leucine-rich repeats are located at the amino terminus and show higher similarity to each other than LRRs in other proteins. In addition to densin three LAP proteins have been found thus far in vertebrates: Erbin (Borg et al., Nat Cell Biol 2:407- 14, 2000), scribble (Nakagawa et al., Mol Cell Biol 20:8244-53, 2000) and LANO (Saito et al., J Biol Chem 276:32051-5, 2001). Densin localizes mainly at the site of cellular synapses as in the dendrites in hippocampal neuronal cultures. Also podocytes are strictly divided into apical and basal compartments along the division plane of the slit diaphragm. Thus, as in other cellular locations densin may be one of the important molecules needed for the maintenance of podocyte polarity. The function of densin in bridging presynaptic to postsynaptic membranes in the glutamatergic synapses (Apperson et al., J Neurosci 16:6839-52, 1996) also remains intriguing: the physical dimensions match closely with the interpodocyte distance separated by the slit diaphragm. Homotypic adhesion found in some LRR containing proteins also resembles the proposed functions of nephrin as an interpodocyte space. Typical function for the type I PDZ domains is binding to distinct C-terminal sequences of other proteins. It is of particular interest that the newly proposed additional component of the slit domain, FAT (Inoue et al, Kidney h t 59:1003-12, 2001) contains typical three C terminal amino acids (TEN) interacting with the PDZ I type domains (Inoue et al., Kidney Int 59:1003-12, 2001; Kim et al., Nature 378:85-8, 1995; Schultz et al., Nat Struct Biol 5:19-24, 1998). Also podocalyxin and megalin contain the same sequence in their C terminus.

The term "densin-like compounds" means "densin" or "densin-like proteins" but also includes nucleic acid sequences encoding said densin or densin-like proteins and binding substances specifically recognizing and binding to or interacting with said densin-like compounds.

The term "densin-like compounds" include polypeptides "substantially homologous" at amino acid level having a significant similarity or identity of at least 80%, preferably 85%, most preferably more than 90% with human forms of densin. The densin-like compounds can also be produced by synthetic, semisynthetic, enzymatic and other biochemical or chemical methods including recombinant DNA techniques. Particularly, they can be produced in cultivated podocytes expressing densin.

The term "densin-like compounds" comprises proteins or polypeptides having the structure, properties and functions characteristic of densin-like proteins, including densin-like proteins, wherein one or more amino acid residues are substituted by another amino acid residue. Also truncated, complexed or chemically substituted, forms of said densin-like proteins are included in the term. The truncated proteins can be truncated in the N-terminal or C-terminal end of the protein, but also in the middle of the protein, e.g. a transmembrane domain can be deleted and provide soluble proteins as well as tryptic peptides as disclosed by Apperson et al. J Neurosci. 16:6839-52, 1996. Chemically substituted forms include for example, alkylated, esterified, etherified or amidized forms with a low substitution degree, especially using small molecules, such as methyl or ethyl, as substituents, as long as the substitution does not disturb the properties and functions of the densin-like proteins. The truncated, complexed and/or substituted variants of said polypeptides can be produced by synthetic or semisynthetic methods, including enzymatic methods and recombinant DNA techniques. The only other prerequisite is that the derivatives still are substantially homologous with and have the properties and/or functions characteristic of densin-like proteins.

The term "densin-like proteins" otherwise covers all possible isoforms or splice variants of densin expressed by different tissues or cells, particularly glomerular podocyte cells. The term "isoform" refers to the different forms of the same protein, which originate from different mammalian, preferably human sources, h the present invention the term, thus, includes fragments, complexes and their derivatives, which can be combined even if they are from different sources. Isoforms of densin-like compounds can be generated by cleavage. Different enzymatic and non-enzymatic reactions, including proteolytic and non-proteolytic reactions, are capable of creating truncated, derivatized or complexed forms of densin-like proteins. They can also be prepared as fusion proteins (Apperson et al. J Neurosci. 16:6839-52, 1996).

The term "properties and/or functions of densin-like proteins" means the capacity of maintaining the cell shape and apical-basal polarity in epithelia of podocytes as well as in presynaptic bridging in synaptic membranes in the slit diaphragm or interpodocyte space in the podocytes.

In the present invention the term "densin-like compounds" includes nucleic acid sequences, which belong to the active densin-like compounds family of the present invention and which comprise isolated or purified "nucleic acid sequences" encoding mammalian, preferably human densin-like proteins or nucleic acid sequences with substantial similarity with said nucleotide sequences, as long as they are capable of encoding amino acid sequences having the essential structural features as well as the properties and/or functions of said densin-like proteins. The nucleotide sequences can be used as such or introduced into suitable transformation or expression vectors, which in turn can be incorporated into the chromosomes of appropriate hosts to provide prokaryotic, eukaryotic organisms, including transgenic animals, e.g. knockout mice capable of expressing altered levels of densin-like proteins. Especially important is the application of the nucleotide sequences for gene therapy. The nucleic acid sequences must be able of encoding densin-like proteins, which are recognizable by binding substances specifically recognizing and binding to and interacting with said densin-like compounds.

The "nucleic acid sequences" of the present invention are not in their natural state, but are isolated and purified from their natural environment as transiently expressed mRNAs. Thereafter, the mRNAs are purified and multiplied in vitro in order to provide by technical means new copies, which are capable of encoding said mammalian densin - like proteins. The nucleic acid sequences include both genomic sequences and cDNA, including their complementary sequences. The term "genomic sequence" means the corresponding sequence present in the nucleus of the mammalian cells and comprises introns as well as exons. In the present context the term "cDNA" means a DNA sequence obtainable by reversed translation of mRNA transcribed from the genomic DNA sequence including its complementary sequence.

The term "nucleic acid sequence encoding densin-like proteins" means nucleic acid sequences encoding densin or substantially homologous sequences. Said sequences or their complementary sequences or nucleic acid sequences containing said sequences or parts thereof, e.g. fragments truncated at the 3'-terminal or 5'-terminal end or in the middle of the sequence, as well as such sequences containing point mutations, are especially useful as probes, primers and for preparing DNA constructs, plasmids and/or vectors useful for modulating the level of expression in the kidney glomeruli. It is, however, clear for those skilled in the art that other nucleic acid sequence are capable of encoding densin-like proteins having the function defined above. Said nucleic acid sequences and/or their complementary sequences should be capable of hybridizing under highly stringent condition (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989). The nucleic acid sequences of the present invention should have a substantial similarity with the nucleic acid sequences encoding densin or densin-like proteins.

"Substantial similarity" in this context means that the nucleotide sequences fulfill the prerequisites defined above and have a significant similarity, i.e. a sequence identity of at least 70 %, preferably more than 80 %, most preferably more than 90 % with said sequences. The "densin-like compounds" of the present invention include densin-like proteins as well as nucleic acid sequences encoding them or binding substances recognizing them, such as ligands and antibodies. Especially important are the applications in which secreted densin-like molecules or fragments thereof or autoantibodies in the subjects in risk of developing or suffering from a glomerular disease are determined with per se known immunochemical methods from biopsy, but preferably from blood, serum and/or urine samples, most preferably from urine samples using antidensin antibodies or densin-like compounds respectively. The densin-like proteins and fragments thereof are also useful as standards in competitive immunoassays, which enable quantification of the results.

As a conclusion "densin-like compounds" in its broadest aspect in the present invention, covers not only densin-like compounds derived from nature, including their isoforms of different origin, but also synthetically, semisynthetically or enzymatically produced densin-like compounds including molecules produced by recombinant DNA techniques, but also the nucleic acid sequence encoding them and "binding substances". Said densin-like compounds can be used either as separate entities or in useful combinations.

Preferably, all densin-like compounds and derivatives thereof should be recognizable using "binding substances" capable of recognizing the natural human densin or densin- like compounds. The term "concentration" or "quantification" includes the determination of the amount of densin-like compounds and is generally determined with binding substances. The quantification of densin-like compounds is preferably determined or screened by per se known immunochemical methods or assays using known amounts of densin-like compounds and/or their respective binding substances as standards. Particularly useful are competitive assays.

The term "binding substances" means substances, which are capable of specifically recognizing and binding to or interacting with natural mammalian, human densin-like compounds or derivatives thereof, preferably to a specific portion thereof. Accordingly, the densin-like compounds include in addition to the proteins and nucleic acid sequences also binding substances. The term "binding substances" means substances, which specifically recognize and bind to or interact with densin-like compounds.

"Binding substances" comprise antibodies, receptors, ligands or specific portions thereof, including any binding proteins or peptides, capable of specifically binding said densin-like compounds, but above all they mean antibodies capable of specifically recognizing one or more densin-like compounds alone or in any combination. The antibodies include both polyclonal and/or monoclonal antibodies as well as fragments or derivatives thereof. Preferably, binding substances recognizing and binding sequence specific epitopes or active sites of the densin-like compounds should be chosen.

Said binding substances can be produced using domains of densin or densin-like compounds, their isomers as well as their fragments, derivatives and complexes which can act as antigens, h other words, antigens include any compositions or materials capable of eliciting an antibody response specific to said densin-like compounds. Said binding substances, preferably antibodies can be produced by conventional techniques for producing polyclonal antibodies as well as monoclonal antibodies. The methods for preparing monoclonal antibodies include hybridoma techniques. Fragments of antibodies or other binding proteins like specific binding peptides can be developed by phage display techniques and produced by recombinant DNA techniques. All methods are well known by those skilled in the art and described in laboratory handbooks.

"Binding substances" are useful especially for diagnosing glomerular diseases. The binding substances are useful for manufacturing means or test kits for demonstrating glomerular diseases in tissue and blood samples but particularly from urine samples.

"Interacting compounds" include above all nephrin and specific antinephrin antibodies. Other "interacting compounds" are for example alpha-actin-4, podocin, P- cadherin, N-cadherin, FAT, NPRAP, delta-katenin or synaptopodin. Alρha-actinin-4 has been directly shown to interact with densin PDZ domain (Walikonis et al., J Neurosci 21:423-33, 2001). This molecule is also important for the cytoarchitecture of podocytes (Smoyer et al., Am J Physiol 273 :F 150-7, 1997) and could be important also for appropriate linking of densin into the cytoskeleton. Interestingly, mutations in the ACTN4 gene lead to the familial focal segmental glomerulosclerosis (FSGC) (Kaplan et al., Nat Genet 24:251-6, 2000). Another interacting partner of densin is (alpha)-subunit of Ca2+/calmodulin-dependent protein kinase II (CaM kinase H) (Apperson et al., J Neurosci 16:6839-52., 1996; Strack et al., J Biol Chem 275:25061-4, 2000; Walikonis et al., J Neurosci 21:423-33, 2001). Thus, interaction has shown to occur also with adherens junction protein delta-catenin/neural plakophilin-related armadillo repeat protein (NPRAP) at synapses where densin-180 has shown to co-immunoprecipitate with delta-catenin/NPRAP and N- cadherin. (Izawa et al., J Biol Chem 277:5345-50, 2002). These interacting compounds also include CD2 associated protein (CD2AP) (Shih et al., Science 286:312-5, 1999), alρha-actinin-4 (Kaplan et al., Nat Genet 24:251- 6, 2000), podocin (Boute et al., Nat Genet 24:349-54, 2000), P-cadherin (Reiser et al., J Am Soc Nephrol 11:1-8, 2000) and FAT (Inoue et al., Kidney it 59:1003-12, 2001). Two of these proteins, CD2AP and podocin, are shown to interact with nephrin (Palmen et al., J Am Soc Nephrol 13:1766-72., 2002; Shih et al, Am J Pathol 159:2303-8, 2001; Yuan et al., Am J Physiol Renal Physiol 282:F585-91, 2002; Huber et al., J Biol Chem 276:41543-6, 2001; Schwarz et al., J Clin Invest 108:1621-9, 2001) maintaining the connection of slit diaphragm to the actin cytoskeleton (Yuan et al., Am J Physiol Renal Physiol 282:F585-91, 2002; Yuan et al., J Am Soc Nephrol 13:946-56, 2002). Another postsynaptic density (PSD) protein, synaptopodin, is similarly shared by the podocytes and the brain with slightly differing molecular weight in these tissues due to glycosylation (Mundel et al., J Cell Biol 139: 193-204, 1997).

"Interacting substances" are useful for screening for compounds which can modulate the interactions in the membranes of kidney and therefore may enable the manufacturing of useful medicines for the treatment of glomerular diseases.

The term "diagnosing" means judging, predicting, assessing or evaluating from in vitro recorded results, if a person is susceptible of or suffers from glomerular diseases or disorders. The diagnostic determinations also enable evaluation of the severity of the condition, the therapy required and the efficacy of treatment modalities, including the medical treatment, especially, when the test is repeated. The diagnostic assay enables early identification of symptoms of the disease and enables prophylactic and/or other treatments before the onset of the actual disease.

The results can be recorded with means for performing immunoassays using densin-like proteins and/or their binding substances as well as parts thereof. The nucleic acid sequences encoding densin-like proteins can be determined with means for carrying out PCR or hybridization techniques using sequence specific probes or primers, which can be selected from the parts of the nucleic acid sequences encoding the specific domains of human densin-like proteins.

The term "immunoassay" refers to immunochemical methods or procedures capable of detecting and/or measuring at least one substance, either a densin-like compound or an antibody recognizing said compounds using per se known means for performing an immunoassay, which means include at least one substance capable of specifically recognizing the substance to be determined, i.e. either at least one binding substance or a densin-like compound or fragments thereof, depending upon the desired application.

Well known examples of immunoassays are radioimmunoassays (RIA), radioimmunometric assays (IRMA), fluoroimmunometric assays (TFMA) enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), fluoroimmmunoassays (FLA), luminescence immunoassays, immunoagglutination assays, turbidimetric immunoassays, nephelometric immunoassays, etc. For quantification competetive assays can be used. All methods are well known by those skilled in the art and described in laboratory handbooks.

The term "prophylactic treatment" includes specific dietary measure and/or precautions, e.g. before the onset of glomerular disease. After the onset of the disease, "prophylactic treatment" requires therapeutic treatment of the disease as a precaution in order to avoid further kidney complications.

The term "therapeutic treatment" includes methods for treating persons by adminstering densin-like compounds including proteins, nucleic acid sequences encoding the same or binding substances specifically recognizing or precipitating with said densin-like compounds expressed in glomerulus or cultivated podocytes. Therapeutic treatment includes gene therapy and preventing genes causing the disease from expressing the gene products causing the diseases. The methods for preventing the manifestation of glomerular diseases includes expression of binding substances, antisense technology and or knockout techniques, etc.

The General Description of the Invention

The primary objective of the present invention was to elaborate methods for diagnosing glomerular diseases. Densin was found to be expressed by glomerular podocytes. This enabled the raising of specific binding substances e.g. antibodies and provide methods and test kits for detecting densin-like compounds in urine. With the method of the present invention it is possible to detect glomerular diseases more precisely.

The present inventors demonstrated the presence of a new molecular component, densin, in the kidney glomerular podocytes. This molecule was demonstrated in the search for new nephrin associated molecules from human glomeruli after precipitation with antinephrin antibodies followed by mass spectrometric identification. Further verification was obtained by detection of the protein with specific antidensin antibodies in the glomeruli and a cultured podocyte cell line. RT-PCR was used to compare the respective mRNA in the kidney and brain. These data together with the limited tissue expression pattern of densin, like that of nephrin, suggest that nephrin and densin are closely associated in the podocytes.

With the recent molecular findings the podocyte is emerging as a key regulatory cell type involved in glomerular damage, but the underlying mechanisms of interacting protein complexes remain poorly understood, hi order to systematically search for additional podocyte molecules interacting with nephrin, glomerular lysates were precipitated with antinephrin antibodies. Further, the proteins of the precipitate were identified with MALDI-TOF mass fingerprint analysis. One of the proteins thus obtained showed identity with densin, a protein originally purified from rat forebrain postsynaptic density fraction and so far shown to be highly brain specific. In the brain, densin localizes reportedly at the synaptic sites of glutaminergic neurites as an adhesion molecule important for maintaining cell shape and the apical-basal polarity.

The expression of densin appeared distinctly in the glomerulus and cultured podocytes by RT-PCR. Further immunoblotting studies with affinity purified antidensin antibodies i revealed a specific band at 185 kDa in brain and cultured podocytes while in human glomerulus densin appeared as a 210 kDa band. Studies for densin localization in the human kidney cortex sections revealed specific staining in the glomeruli in a podocyte- like pattern while faint reactivity was found beneath the brush border membrane of proximal tubuli as well.

Due to its known involvement in the synaptic organization, maintenance of cell shape and polarity in nerve cells, together with its shown interactions with alpha-actinin-4, densin may share the same functions in podocytes by associating with the nephrin interacting protein complex at the slit diaphragm.

In order to systematically screen for additional molecules interacting with nephrin, the present inventors performed coprecipitation experiments with glomerular lysate using a nephrin spesific antibody and analyzed the proteins precipitated with mass spectrometry. Surprisingly the inventors found a protein, densin, previously only found in the central nervous system. Densin belongs to a family of proteins postulated to have function in cell adhesion and polarity. The inventors verified the densin expression in glomerular podocytes by RT-PCR, immunofluorescence, western blotting and immunoelectron microscopy. The results of this study indicate that densin may be one component of a larger complex of functional proteins localized to the slit diaphragm area with a possible role in glomerular filtration. Human glomerular lysate imrnunoprecipitated with antinephrin antibody #1109 showing coprecipitation of densin is depicted in Figure 2B. Such precipitation with a control sample, Protein A sepharose 4B, cannot be seen.

Imrnunoprecipitated samples from rat glomeruli were used for the mass analysis, MALDI-TOF-MS (see Example 7). A list of peptide masses obtained in MALDI-TOF- MS analysis was used to search the NCBI database using ProFound software (Version 4.10.5, Rockefeller University). 11 of the peptide masses were found to match to rat densin covering 10% of 1495 long amino acid sequence.

The present inventors have now shown that densin is expressed also in glomerular epithelial cells, podocytes. Densin expression from cultured human podocytes, human kidney cortex and human glomerulus (Figure 1) was shown with reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Therefore densin-like compounds can be produced in sufficient amounts using cultivated podocytes. Also recombinant DNA techniques can be used for the production. The primers comprising the sense primer 2485U (SEQ ID NO:6) and the anti-sense primer 2692L (SEQ ID NO:7) designed according to the rat densin sequence (GenBank accession number U66707) were used in PCR. The same primers were used for human and rat cDNA due to only two base difference inside the primer regions with no changes at 3' end of the primers. RT-PCR analysis revealed densin mRNA expression in glomeruli as well as in cultured human podocytes (Figure 1). Amplification of the cDNA prepared from rat brain tissue gave a band of the same size when the samples were run in 1.5% agarose gel. Results were confirmed by sequencing the amplified PCR products of brain and podocytes revealing 100 % identity with densin.

In order to further characterize the densin protein found in the glomerular podocytes an immunoblot of cell and tissue lysates from human brain, cultured podocytes and glomerular lysates was performed (Figure 2A). Analysis with the affinity purified polyclonal rabbit anti-densin antibody directed to the putative extracellular domain of human densin identified a protein band with an approximate molecular weight of 185 kDa in the brain lysate and the human podocytes, whereas 210 kDa band was detected in the human glomeruli lysate. A 185 kDa band as was described earlier in rat brain lysate (Izawa et al., J Biol Chem 277:5345-50, 2002).

In addition to antibodies, receptors or binding proteins or peptides, capable of specifically binding to densin-like molecules can be used, but above all binding substance antibodies capable of specifically recognizing one or more densin-like compounds alone or in any combination. The antibodies include both polyclonal and/or monoclonal antibodies as well as fragments. Preferably, the binding substances recognizing and binding to densin-like compounds should be chosen from specific epitopes or active sites of the densin-like molecules should be chosen.

Binding substances such as monoclonal and polyclonal antibodies can be produced using densin or any densin-like compounds, their isomers as well as their fragments, derivatives and complexes with the prerequisite that they are capable of acting as "antigens", in other words, antigens include any compositions or materials capable of eliciting an antibody response specific to said densin-like compounds. Said binding substances, preferably antibodies are producible by conventional techniques for producing polyclonal antibodies as well as monoclonal antibodies. The methods for preparing monoclonal antibodies include hybridoma techniques. Fragments of antibodies or other binding proteins like specific binding peptides can be developed by phage display techniques and produced by recombinant DNA techniques. All methods are well known by those skilled in the art and described in laboratory handbooks.

Immunoblotting of human urine samples from type I diabetic patients with the affinity purified densin antibody is presented in Figure 4. The urine samples from patients with normal albumin excretion (patients without glomerular damage) and from patients with severe albuminuria (patients with prolonged glomerular disease) were compared. It was seen that densin was detected in urine of normoalbuminuric patients, whereas after the occunence of glomerular disease densin is not found in urine. This shows that densin can be used as a marker in diagnostics of glomerular diseases. An abenant amount of densin in a subject presenting with proteinuria or in the risk of developing proteinuria indicates a predisposition or higher risk of a glomerular disease.

Immunofluorescence staining of frozen sections of normal human or CNF kidney cortical tissue samples with the affinity purified densin antibodies revealed intensive glomerular staining in normal human glomeruli (Figure 3A). Furthermore, the fluorescence on 1 micron thick frozen sections of formalin fixed normal human kidney was clearly observed (Figure 3B). The antibody labels podocytes and the base of microvilli of proximal tubules.

When semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the differences in transcript levels in the proteinuric hereditary congenital nephropathy of the Finnish type (CNF) kidneys, increased densin mRNA expression was seen in CNF kidney cortex (Figure 11 A). These results suggest that the quantitation of densin expression levels can be used in diagnosing kidney diseases.

The results here show densin mRNA and protein in the kidney glomerulus and cultured podocytes. According to the amino acid sequence the calculated molecular weight of densin is 167 kDa. However, in immunoblotting experiments the size of densin appeared to be 185 kDa in the human and rat brain as well as in human podocytes using affinity purified antidensin antibodies. In the human glomerulus the molecular size of densin, however, was 210 kDa. The differences in the molecular weights observed are most likely due to tissue-specific posttranslational modifications since densin belongs to the O-sialoglycoprotein family abundant in sialic acids. (Apperson et al., J Neurosci 16:6839-52, 1996). Another postsynaptic density (PSD) protein, synaptopodin, is similarly shared by the podocytes and the brain with slightly differing molecular weight in these tissues due to glycosylation (Mundel et al., J Cell Biol 139: 193-204, 1997). In immunofluorescence studies anti-densin antibody shows strong reactivity in podocytes and weakly also in the proximal tubular brush border area. Whether the observed tubular expression reflects true in situ functional densin or whether it is due to leakage of soluble densin into urine much alike nephrin (Aaltonen et al., Lab Invest 81:1185-90, 2001) and followed by tubular reabsorbtion remains to be studied. Immunoblotting of human urine samples from diabetic patients with the affinity purified densin antibody is presented in Figure 4. The urine samples from normoalbuminuric and highly proteinuric patients were compared. It was seen that densin was detected in urine of healthy patients, whereas after the occunence of glomerular disorder densin was not found in urine.

When urine samples from 25 type I diabetic patients with normal albumin excretion and from 25 normal normal controls were compared densin was detected in the urines of 9 normoalbuminuric diabetic patients (9/25) whereas only 3 control subjects (3/25) showed densin immunoreactivity. This shows that densin can be used to predict the risk or presence of kidney disease in diabetic patients and densin is a possible marker in diagnostics of glomerular diseases. Urinary densin is further imrnunoprecipitated with affinity purified anti-densin antibody, the precipitated samples are separated on SDS-PAGE gels and silver stained. A stained protein band is excised and further processed for analysis. The identification of the protein is performed with mass spectrometry.

Patients with type I diabetes mellitus typically develop autoantibodies. Since densin is a novel protein expressed in the kidney as well as in the pancreas it is useful to test whether densin autoantibodies are found in these patients. Patients with type I diabetes and healthy control subjects are analyzed for densin autoantibodies using a radioimmunoprecipitation assay, Western blotting, ELISA or another standard in vitro diagnostic method. The sera to be analyzed is taken at the time of diagnosis of diabetes and after a certain follow-up period. The proportion of patients having densin autoantibodies at the time of diagnosis and in each follow-up sample is determined, and these results are analyzed together with the clinical data to study whether densin autoantibodies are related to the predisposition, pathogenesis or progression of type I diabetes and/or diabetic nephropathy.

The transmembrane nature of nephrin with an extracellular and a characteristic intracellular domain has shown to reflect functionality. The extracellular domain most likely mediates outside in signaling (Simons et al., Am J Pathol 159:1069-77, 2001). The intracellular part of nephrin has no marked similarity to other known proteins and according to recent evidence mediates binding to the cytoskeleton via CD2AP (Palmen et al., J Am Soc Nephrol 13:1766-72, 2002; Shih et al., Am J Pathol 159:2303-8, 2001). Association with podocin (Schwarz et al., J Clin Invest 108:1621-9, 2001) suggests that the many cytoplasmic proteins of podocytes converge to form the slit diaphragm with its nephrin backbone (Tryggvason et al., Int J Dev Biol 43:445-51, 1999). This kind of compartmentalization to functional domains has been proposed previously (Kerjaschki, J Clin Invest 108:1583-7, 2001) and is directly supported by our recent findings (Luimula et al., Lab Invest 82:713-8, 2002). Nephrin association preferentially to slit diaphragm and particularly in the lipid rafts like many proteins of the central nervous and immunologic systems may suggest of some shared functions. Here we did not, however, find densin association in the lipid but, instead in the heavy fractions where the main pool of nephrin is also seen (data not shown). The absence of densin in the lipid rafts raises the question whether the interaction between nephrin and densin postulated here occur due to a larger protein complex or is it a direct interaction with the non lipid raft associated form of nephrin. The first is supported by the fact that in the lysate most of the membrane proteins are still insoluble and larger membrane complexes remains in the supernatant allowing precipitation of proteins without direct interactions. However, the second option is not excluded while it is known that low detergent preserve most of the protein-protein interactions. More over Strack et al., (J Biol Chem 275:25061-4, 2000) have suggested that similar to nephrin (Holthofer et al., Am J Pathol 155:1681-7, 1999; Ahola et al., Am J Pathol 155:907-13, 1999; Aaltonen et al., Lab Invest 81:1185-90, 2001; Luimula et al., Pediatr Res 48:759-62, 2000; Saleem et al., J Am Soc Nephrol 13:630-8, 2002), densin shows differently spliced isoforms with typical expression patterns during development and apparently also with different functional states.

The results demonstrate that densin is an important molecule for the function of glomerulus and herein it is suggested as a new target molecule for preparing therapeutic and diagnostic products including antibodies facilitating glomerular disease diagnostics.

The invention is described in more detail in the following examples.

Example 1 Tissue samples

Cadaver kidneys unsuitable for transplantation for vascular anatomic reasons (Department of Surgery, University of Helsinki) or the normal poles of kidneys removed because of Wilms' tumor were used. The cortical tissue was separated from the medulla and was immediately frozen in liquid nitrogen and stored at -70°C until processed for RNA isolation, immunohistochemistry or electron microscopy as described below. Glomeruli were isolated with the sieving method as described earlier (Holthofer et al., Kidney Int 45:123-30, 1994). The human brain sample was from normal tissue of young adult's temporal lobe removed during tumor surgery. Example 2 Cell culture

Human podocytes were isolated from normal human kidney as earlier described (Greiber et al., Kidney hit 53:654-63, 1998; Huber et al., J Immunol 168:6244-52, 2002). Briefly, isolated glomeruli were suspended in DMEM containing 10% heat- inactivated FCS, 2.5 mM glutamine, 0.1 mM sodium pyruvate, 5 mM HEPES buffer, 1 mg/ml streptomycin, 100 U/ml penicillin, O.lx nonessential amino acids (lOOx; all Seromed, Berlin, Germany), insulin, transferrin, and a 5 mM sodium selenite supplement and were incubated at 37°C and 5% CO in air. Cell colonies sprouted around the glomerula were excised and incubated in 5 ml 0.2% collagenase IV (Sigma- Aldrich, Deisenhofen, Germany) at 37°C for 30 min followed by washes and further plating. Cells showed an epithelial morphology and were stained positive for Wilm's tumor antigen (WT1) and nephrin, markers which are only found in podocytes in the adult kidney. Cells were also negative for endothehal cell marker, factor 8 related antigen.

Example 3

Production of Antibodies

The whole intracellular sequence of rat nephrin (GenBank AF 125521; SEQ ID NO:l) was cloned to pGEX-2T expression vector (Amersham Pharmacia Biotech, Upsala, Sweden) and transformed into the E.coli strain BL-21 (Amersham Pharmacia Biotech) followed by culture in 2 x YTAG medium (yeast extract 10 g/L, tryptone 16 g/L, NaCl 5g/L, glucose 2% and 100 μg/mL ampicillin) at 37°C. After isopropyl beta-D- thiogalactosidase (IPTG) (Amersham Pharmacia Biotech) induction expression was allowed for 1-2 hours before purification as described earlier (Frangioni et al., Anal Biochem 210:179-87, 1993). Shortly, the cells were lysed in STE buffer (150 mM NaCl, 10 mM Tris pH 7.5 and 1 mM EDTA) at the presence of 100 μg/ml lysozyme, 3% sarcosyl and 5 mM dithiothreitol following clearing with centrifugation at 7000 x G. Supernatant was adjusted to 2% with Triton X-100 before fusion protein collection with glutathione sepharose 4B (Amersham Pharmacia Biotech). Eluted protein was used for immunizations. The respective number for the antiserum produced with this antigen is #050.

The human nephrin sequence 3091 - 3645 (numbered according to Genbank AF 035835; SEQ ID NO:2) lacking the exon area 24 (AF126957; SEQ ID NO:3) was first inserted into donor vector pUni V5-His-TOPO (rnvitrogen, Carlsbad, CA) followed by fusion to acceptor vector pBAD/Thio-E Echo (rnvitrogen) according to manufacturer's protocol. This thioredoxin fusion construct was transformed to TOP 10 E. coli strain for further culturing in LB medium with added kanamycin 50 μg/mL. Expression was induced with 0.02% arabinose followed by production at 37°C for 4 hours. Collected cells were lysed with 100 μg/mL lysozyme, sonication and flash freezing in the presence of the Complete Mini EDTA-free protease inhibitor mix (Roche, Mannheim, Germany). Purification was performed with HisTrap Kit (Amersham Pharmacia Biotech) using nickel as binding cation for coupling the fusion protein. Bound protein was eluted using a imidazole step gradient from 40 to 300 mM. Antiserum against this human antigen used is number #1109.

To obtain antiserum rabbits were immunized with standard protocol as earlier described (Holthofer et al., Am J Pathol 155:1681-7, 1999). Briefly, the suitable antigen was injected in Freud's complete adjuvant (Difco Laboratories, Detroit, MI) in multiple sites in the back and appropriate booster immunizations were given at 4 weeks intervals. The serum was collected and tested for specificity.

Production and characterization of the nephrin antipeptide antibody #409 has been earlier described in detail (Holthofer et al., Am J Pathol 155:1681-7, 1999; Ahola et al., Am J Pathol 155:907-13, 1999). Basically, intracellular peptide covering amino acids 1101 -1126 was produced in The Peptide Synthesis Unit of Haartman institute, University of Helsinki followed by immunizations to rabbits as described above. Specificity was tested with immunoblotting, immunofluorescence and immunoelectron microscopy. Moreover, immunoprecipitation procedure was performed by in vitro transcription and translation of full-length nephrin sequences. Characterization of nephrin antibodies

Antiserums #050 and #1109 were tested for specificity to nephrin by immunoblotting and immunoprecipitation using lysates of isolated glomeruli. Further preincubation of #050 with the conesponding antigen used for immunizations blocked blotting reaction. Antibodies were also tested on kidney cortical sections with immunofluorescence assay revealing distinct reactivity exclusively with glomeruli.

Example 4

Production of densin antibodies

Production and characterization of the polyclonal, affinity purified, anti-densin antibody directed to the extracellular part of densin has been described earlier (Izawa et al., J Biol Chem 277:5345-50, 2002). Briefly, (His₆)-tagged recombinant proteins with the putative extracellular amino acids 475-754 (according to human densin sequence AF 434715; SEQ ID NO:4) were produced and injected to rabbits. The antiserum was affinity purified using the same amino acid region produced as GST- fusion protein. Specificity of the antibody was shown against rat brain densin using the Western blot method.

Example 5 Immunoprecipitations

Normal rat or human glomerular lysates used in the immunoprecipitation studies were prepared using the same lysis buffer as for isolation of detergent-resistant membranes (DRMs) known to preserve lipid rafts. (Schwarz et al., J Clin Invest 108:1621-9, 2001; Simons et al., Am J Pathol 159:1069-77, 2001). Briefly, isolated glomeruli from the rat or human kidney were homogenized in TNE buffer (250 mM NaCl, 5 mM EDTA, 10 mM Tris, pH 7.4) with 0.2% Triton X-100 at +4°C. The resulting homogenate was centrifuged and the pellet obtained was homogenized in TNE buffer similarly once more. Supernatant from the first centrifugation and the second pellet homogenate were pooled and passed through a 25 gauge needle. After 45 min incubation on ice the lysate was centrifuged at 14000 rpm in an Eppendorf tube centrifuge following supernatant preclearing by Protein A Sepharose 4B (Zymed, San Francisco, CA) for 1 hour at +4°C. The beads were collected and 20μl anti-nephrin antiserum was added. After over night incubation at +4°C Protein A Sepharose 4B was added followed by collection and washes with homogenization buffer. Finally, Laemmli sample buffer was added to washed beads and the mixture was cooked for 10 min at 100°C.

Coimmunoprecipitations

After precipitation with nephrin antibody #1109 from human glomerular lysate the resulting presipitate was separated on SDS PAGE followed by Western blotting using affinity purified antidensin as primary antiboby. This revealed densin band of size 210 kDa while no such band was observed from glomerular lysate incubated with Protein A Sepharose 4B only (Figure 2B).

Example 6

Sample preparation for MALDI-TOF-MS analysis

Imrnunoprecipitated samples from rat glomeruli were suspended in reducing Laemmli buffer (62.5mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.05% bromophenol blue) followed by separation on 8% SDS-PAGE -gels and silver staining according to Blum et al (Electrophoresis 8:93-99, 1987). A stained protein band with the approximate size of 200 kDa was excised from the gel and further cut into pieces as described by O'Connel and Stults (Electrophoresis 18:349-59, 1997) Briefly, the stain was removed with 0.2 M NH₄HCO₃/ACN solution 1:1 at 37°C for 45 min followed by additional ACN treatment and drying in vacuum centrifugation. Disulfide bonds were blocked using 55 mM iodoacetamide in 0.1 M NH₄HCO₃ at room temperature in the dark. The gel pieces were washed using 0.1 M NH₄HCO and H₂O with ACN shrinking between and final drying in vacuum. Trypsin digestion was performed over night at 37°C with 0.05 μg/μl sequencing grade modified trypsin (Promega, Madison, USA) followed by peptide extraction from the gel by 0.1% TFA/ 60% ACN 30 min at 37°C. After additional 0.1% TFA treatment, peptides were desalted and saturated with equal volume of β-cyano4-hydroxy cinnamic acid. This mixture was used for the mass analysis (see below Example 7).

Example 7 MALDI-TOF-MS analysis

Fingerprinting for trypsined peptides was performed with a Biflex™ matrix-assisted laser desoφtion/ionization-time of flight (MALDI-TOF) mass spectrometry (2GHZ digitizer) (Bruker, Rheinstetten, Germany) at the Protein Chemistry Unit at Biomedicum, Umversity of Helsinki. Positive ion reflector mode was used with an accelerating voltage of 19 000 V and delayed extraction of 2 ns. Internal peptide calibration standards (Bruker Daltons, Bremen, Germany) were applied to obtain higher peptide mass accuracy.

The list of peptide masses obtained in MALDI-TOF-MS analysis was used to search the NCBI database using ProFound software (Version 4.10.5, Rockefeller University). 11 of the peptide masses were found to match to rat densin covering 10% of 1495 long amino acid sequence.

Example 8 RT-PCR

Total RNA was extracted from the cultured cells or isolated tissues using Trizol® reagent (Life Technologies, Gibco BRL, Paisley, Scotland) according to manufacturer's instructions. RNase free DNase I (Promega, Madison, WI) together with human placental RNase inhibitor (Promega) was used to remove genomic DNA. cDNA synthesis was carried out with the Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT) enzyme (Promega) in the presence of an oligo dT 15 -primer and an RNase inhibitor.

PCR for densin cDNA was performed with sense 2485U 5'-atgctttccctgacaactgg-3' (SEQ ID NO:6), antisense 2692L 5'-gtgtgtctgtgggtggactg-3'(SEQ ID NO:7) primers designed according to the rat densin sequence (GenBank accession number U66707; SEQ ID NO: 5). The same primers were used for human and rat cDNA due to only two base difference inside the primer regions with no changes at 3' end of the primers. Amplification program included denaturation at 94°C followed by 35 cycles with 30 s at 94°C, 30 s at 56°C and 30 s at 72°C. After the last cycle elongation at 72°C for 10 min was allowed.

RT-PCR analysis revealed densin mRNA expression in glomeruli as well as in cultured human podocytes (Figure 1). PCR amplification of the cDNA prepared from rat brain tissue gave a band of the same size when the samples were run in 1.5% agarose gel. Thr results were confirmed by sequencing the amplified PCR products of brain and podocytes revealing 100% identity with densin.

Example 9 Immunoblotting

For SDS-PAGE, the detergent extracts of tissue samples were suspended in reducing Laemmli buffer and heat denaturated at 100 °C for 10 minutes. Proteins were electrophoresed in 8% sodium dodecyl sulphate (SDS) polyacrylamide gel under reducing conditions and electrotransfened onto a nitrocellulose membrane (Amersham Life Science, Buckingham, England). Unspecific binding was blocked by incubating in 3% bovine serum albumin (INC Biomedicals, Aurora, Ohio). The primary and secondary antibodies were diluted to the same blocking reagent. Affinity purified polyclonal rabbit anti-densin antibody directed to the putative extracellular part of densin was used at 1:1000 dilution followed by an incubation with the horseradish peroxidase -conjugated affiniPure goat anti-rabbit IgG (H+L) (Jackson nmuno Research Laboratories Inc., West Grove, PA), at 1:50000. All washes were done with phosphate buffer saline (PBS). Visualization of the bound antibodies were done using SuρerSignal®West Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL).

In immunoblotting experiments the appropriate cell and tissue lysates were blotted with affinity purified anti-densin antibodies raised against the extracellular part of human densin (Figure 2A). These experiments revealed the typical 210 kDa band detected in human glomerli lysate while a 185 kDa band was obtained in the lysate of human podocytes. Also, blotting with human brain lysate revealed the samel 85 kDa band as was described earlier in rat brain lysate (Izawa et al., J Biol Chem 277:5345-50, 2002).

Example 10 Immunofluorescence

Normal human or CNF kidney cortical tissue samples were prepared for immunofluorescence as described earlier (Holthofer et al., Faseb J 13:523-32, 1999). Briefly, 5 μm frozen cortical sections were fixed in acetone at -20°C for 10 minutes and incubated with the primary antibodies diluted in 10% normal human serum at +4°C overnight. The affinity purified rhodamine (TRITC)- conjugated goat anti-rabbit Ig (Jackson hnmuno Research Laboratories Inc., West Grove, PA) was used as secondary antibody. Immu-Mount embedding medium (Shandon, Pittsburgh, PA) covered sections were analysed with an Olympus OX50 fluorescence microscope (Olympus Optical GmbH, Hamburg, Germany).

Immunofluorescence studies revealed intensive glomerular staining in normal human glomeruli (Figure 3 A). Furthermore, the good fluorescence on 1 micron thick frozen sections of formalin fixed normal human kidney was observed (Figure 3B). The antibody labels podocytes and the base of microvilli of proximal tubules.

Example 11 Immunoelectron microscopy

Immunoelectron microscopy was done as described earlier (Kerjaschki et al., J Clin Invest 100:2303-9, 1997). After fixation of normal cortex in 4% formaldehyde and embedding with Lowicryl K4M (Chemische Werke LOWl,Waldkraiburg, Germany) 1 micron thick sections were done. These sections were blocked with ovalbumin followed by incubation with affinity purified antidensin antibodies and the relevant 10-nm gold conjugate (1:50). Cryo EM was performed with the same antibodies. Immunoelectronmicroscopy with antidensin antibody shows specific localization at the slit diaphragm domains (Figure 10). Example 12

Demonstration of an aberrant amount of densin in urine samples from patients presenting with proteinuria

24-hour urine collections were obtained from type I diabetic patients with normal albumin excretion (n=2) and with macroalbuminuria (n=2). A volume containing equal amount of total proteins was precipitated from each sample (30 μg) and analyzed with Western blotting using affinity purified densin antibody. Sample preparation, electrophoresis and detection were carried out as described in Example 9.

The results presented in Figure 4 were compared. It was seen that densin was detected in urine of normoalbuminuric patients, whereas after occunence of a glomerular disorder (macroalbuminuria), densin was not found in urine. This shows that densin in urine can be used to predict the risk of glomerular disorders or presence of glomerular disease.

Example 13

Densin is found in the urine samples from diabetic patients

24-hour urine collections were obtained from type I diabetic patients with normal albumin excretion (n=25) and from normal controls (n=25). A volume containing equal amount of total proteins was precipitated from each sample (30 μg) and analyzed with Western blotting using affinity purified densin antibody. Sample preparation, electrophoresis and detection were carried out as described in Example 9.

Densin was detected in the urines of 9 normoalbuminuric diabetic patients (9/25) whereas only 3 control subjects (3/25) showed densin immunoreactivity. This shows that densin in urine can be used to predict the risk or presence of kidney disease in diabetic patients.

Urinary densin is imrnunoprecipitated with affinity purified anti-densin antibody, the precipitated samples are separated on SDS-PAGE gels and silver stained. A stained protein band is excised and further processed for analysis. The identification of the protein is performed with mass spectrometry.

Example 14

Latex agglutination for demonstrating risk of glomerular disease

Urine samples from patients will be collected and brought in contact with latex particles covered with monoclonal antibodies specifically recognizing densin. The lack of or the formation on an agglutination can be used to predict the risk of glomerular disorders or presence of glomerular disease.

Example 15

Semiquantitative RT-PCR and Western blotting

Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the differences in transcript levels in the proteinuric hereditary congenital nephropathy of the Finnish type (CNF) kidneys. Briefly, the 10 μg DNase-treated RNA was reverse transcribed in the presence of an oligo dT15-primer (Boehringer Mannheim), RNase inhibitor, and Moloney murine leukemia virus reverse transcriptase (M-MLN RT) enzyme (Promega). cDΝAs were diluted 1:5 to 1:3125 to find the linear range of PCR reactions. The dilutions of 1:125 to 1:3125 were used in equalizing the amounts of cDΝA according to the /3-actin levels. For semiquantitation of densin, the dilutions of 1:5 to 1:125 were used. Increased densin mRΝA expression was seen in CΝF kidney cortex.

Semiquantitation of densin protein levels was done as described in the Example 9 and in the publication Luimula et al., Lab Invest 82:713-718, 2002. Increased densin protein expression was detected in CΝF glomeruli (Figure 11). These results suggest that the quantitation of densin expression levels can be used in diagnosing kidney diseases.

Example 16

Densin autoantibodies in diabetes and diabetic nephropathy

Claims

1. Use of densin-like compounds or fragments thereof as well as nucleic acids encoding said densin-like compounds or fragments present in the kidney for manufacturing products useful for diagnosing and treating glomerular diseases.

2. The use according to claim 1, characterized in that the densin-like compounds and fragments thereof are expressed by glomerular podocytes and are obtainable from kidney by coprecipitation with antibodies recognizing nephrin and subsequent purification, by expression in cultivated glomerular podocytes or by conventional recombinant DNA techniques.

3. The use according to claim 1, characterized in that the densin-like compounds comprise densin protein, densin-like proteins, fragments or derivatives thereof, nucleic acid sequences encoding densin-like proteins or fragments thereof or binding substances or fragments thereof capable of specifically recognizing and binding to at least one of said densin-like compounds.

4. The use according to claim 3, characterized in that the binding substances specifically recognizing or interacting with densin-like compounds are obtained by producing polyclonal or monoclonal antibodies against said densin-like compounds by per se known methods.

5. The use according to claim 4, characterized in that the binding substances interacting with densin-like compounds are nephrin, CD2 associated protein, alpha- actin-4, podocin, P-cadherin, N-cadherin, FAT, NPRAP, delta-katenin, megalin or synaptopodin.

6. The use according to claim 1, characterized in that the glomerular diseases comprise glomerular diseases manifesting with proteinuria due to hereditary, infectious, inflammatory, toxic or metabolic reasons including but not being restricted to IgA nephropathy, membranous, diabetic, crescentic and minimal change nephropathies as well as nephropathy associated with hypertension and infections including HIV associated nephropathy.

7. The use according to claim 1, characterized in that the glomerular disease comprises diabetic nephropathy.

8. The use according to claim 1, characterized in that the product manufactured using densin-like compound is a nucleic acid sequence encoding functionally active densin- like compounds, a DNA construct, a vector, a plasmid or a cell carrying said nucleic acid sequence.

9. The use according to claim 1, characterized in that the product is a densin-like product or a binding substance specifically recognizing and binding to a densin-like compound in combination with means for carrying out an immunochemical reaction.

10. The use according to claim 1, characterized in that the product is a densin-like product or a binding substance specifically recognizing and interacting with a densin- like compound in combination with means for demonstrating the interaction.

11. A method for in vitro determination of the predisposition or risk of developing a glomerular disease as well as the progress of healing, characterized in that the method comprises contacting a binding substance specifically recognizing and binding to a densin-like protein, a fragment or derivative thereof with a tissue, blood, serum or urine sample from a subject in the risk of developing proteinuria or presenting with proteinuria and recording an amount of densin in the sample.

12. A method for in vitro diagnosing the presence, predisposition or risk of developing a glomerular disease as well as the progress of healing, characterized in that the method comprises contacting a densin-like protein, fragment or derivative thereof with a serum or blood sample from a subject presenting with proteinuria, wherein an immunochemical reaction indicates the presence of autoantibodies to densin.

13. The method according to claim 12, characterized in that the presence of autoantibodies indicates a predisposition or risk of an immunologic disorder causing glomerular disease.

14. A method for in vitro diagnosing the presence, predisposition or risk of developing a glomerular disease as well as progress of healing, characterized in that the method comprises contacting a tissue sample obtained from a kidney biopsy containing nucleic acid sequences from a subject suspected of suffering from a glomerular disease with a probe comprising a nucleic acid sequence encoding a densin-like protein or a fragment thereof, recording a hybridization signal of an amplification product wherein a non- physiological amount of hybridization signal of amplification products indicates a predisposition or risk of glomerular disease

15. A test kit according to the method of any one of claims 11-14 for in vitro diagnosing the presence, predisposition or risk of developing a glomerular disease as well as progress of healing, characterized in that the test kit comprises a densin-like protein, fragments or derivatives thereof in combination with means for performing an immunochemical assay.

16. A test kit according to the method of any one of claims 11-14 for in vitro determination of the amount of a densin-like compound in a sample, characterized in that said test kit comprises at least one binding substance specifically recognizing and binding to a densin-like protein or fragments thereof in combination with means for performing an immunochemical assay.

17. A test kit according to the method of any one of claims 11-14 for in vitro determination of the amount of a densin-like compound in a sample, characterized in that said test kit comprises at least one probe, which is a nucleic acid sequence encoding a densin-like protein or fragment thereof in combination with means enabling a hybridization or a PCR reaction.

18. A method for screening a library of substances for identifying compounds potentially useful for treating glomerular diseases, characterized in that the substances are screened for their capacity of modulating, stimulating respective inhibiting or down- regulating, the interaction between densin-like compounds and nephrin, CD2 associated protein, alpha-actin-4, podocin, P-cadherin, N-cadherin, FAT, NPRAP, delta-katenin, megalin or synaptopodin.

19. A method for treating glomerular diseases, characterized in that the method comprises administering a densin-like protein, fragment or derivative thereof to a subject suffering from glomerular diseases

20. A method for treating glomerular diseases, characterized in that the method comprises administering a binding substance specifically recognizing and binding to a densin-like protein or fragment thereof to a subject suffering from glomerular diseases.

21. A method for treating glomerular diseases characterized in that the method comprises administering a nucleic acid sequence encoding a densin-like protein or fragment thereof to a subject suffering from glomerular diseases.