This invention was made with partial assistance from grant Nos. AG 8245 and DK 19655 from the National

Institutes of Health. The government may have certain rights in this invention.

RELATED PUBLICATIONS

The Applicants are co-authors of the following articles directed to the subject matter of the present invention: "FUNCTION OF MACROPHAGE RECEPTOR FOR NONENZYMATICALLY GLYCOSYLATED PROTEINS IS MODULATED BY INSULIN LEVELS", Vlassara, Brownlee and Cerami, DIABETES (1986), Vol. 35 Supp. 1, Page 13a; "ACCUMULATION OF DIABETIC RAT PERIPHERAL NERVE MYELIN BY MACROPHAGES INCREASES WITH THE PRESENCE OF ADVANCED GLYCOSYLATION ENDPRODUCTS", Vlassara, H., Brownlee, M. , and Cerami, A. J. EXP. MED. (1984), Vol. 160, pp. 197-207; "RECOGNITION AND UPTAKE OF HUMAN DIABETIC PERIPHERAL NERVE MYELIN BY MACROPHAGES", Vlassara, H. , Brownlee, M. , and Cerami, A. DIABETES (1985), Vol. 34, No. 6, pp. 553-557; "HIGH-AFFINITY- RECEPTOR-MEDIATED UPTAKE AND DEGRADATION OF GLUCOSE- MODIFIED PROTEINS: A POTENTIAL MECHANISM FOR THE REMOVAL OF SENESCENT MACROMOLECULES", Vlassara H. , Brownlee, M. , and Cerami, A., PROC. NATL. ACAD. SCI. U.S.A. (Sept. 1985), Vol. 82, pp. 5588-5592; "NOVEL MACROPHAGE RECEPTOR FOR GLUCOSE-MODIFIED PROTEINS IS DISTINCT FROM PREVIOUSLY DESCRIBED SCAVENGER RECEPTORS", Vlassara, H. , Brownlee, M., and Cerami, A. JOUR. EXP. MED. (1986), Vol. 164, pp. 1301-1309; "ROLE OF NONENZYMATIC GLYCOSYLATION IN ATHEROGENESIS", Cerami, A., Vlassara, H. , and Brownlee, M., JOURNAL OF CELLULAR BIOCHEMISTRY (1986), Vol. 30, pp. 111-120; "CHARACTERIZATION OF A SOLUBILIZED CELL SURFACE BINDING PROTEIN ON MACROPHAGES SPECIFIC FOR PROTEINS MODIFIED NONENZYMATICALLY BY ADVANCED GLYCOSYLATION END PRODUCTS", Radoff, S. , Vlassara, H. and Cerami, A., ARCH. BIOCHEM. BIOPHYS (1988), Vol. 263, No. 2, pp. 418-423;

"ISOLATION OF A SURFACE BINDING PROTEIN SPECIFIC FOR ADVANCED GLYCOSYLATION ENDPRODUCTS FROM THE MURINE MACROPHAGE-DERIVED CELL LINE RAW 264.7", Radoff, S., Vlassara, H. , and Cerami, A., DIABETES, (1990), Vol. 39, pp. 1510-1518; "TWO NOVEL RAT LIVER MEMBRANE PROTEINS THAT BIND ADVANCED GLYCOSYLATION ENDPRODUCTS: RELATIONSHIP TO MACROPHAGE RECEPTOR FOR GLUCOSE-MODIFIED PROTEINS", Yang, Z., Makita, Z., Horii, Y. , Brunelle, S., Cerami, A., Sehajpal, P., Suthanthiran, M. and Vlassara, H., J. EXP. MED., (In Press) . All of the foregoing publications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to the nonenzymatic glycosylation of proteins, and particularly to the discovery of binding partners to advanced glycosylation endproducts such as AGE receptors, that may serve in the diagnosis and treatment of conditions in which the presence or activity of such advanced glycosylation endproducts may be implicated.

Glucose and other reducing sugars attach non- enzymatically to the amino groups of proteins in a concentration-dependent manner. Over time, these initial Amadori adducts undergo further rearrangements, dehydrations and cross-linking with other proteins to accumulate as a family of complex structures which are referred to as Advanced Glycosylation Endproducts (AGEs) . Although this chemistry has been studied by food chemists for many years, it was only in the past decade that the presence of AGEs in living tissues has been established. The excessive deposition of these products on structural body proteins as a function of age and elevated glucose concentration, taken together with evidence of effective prevention of tissue pathology by an AGE inhibitor, aminoguanidine, has lent support to the hypothesis that

the formation of AGEs plays a role in the long term complications of aging and diabetes.

Since the amount of AGEs found in human tissues is less than could be predicted from protein/glucose incubation studies in vitro, the applicants herein proposed several years ago that there might be normal mechanisms to remove those long-lived proteins which had accumulated AGEs in vivo. Particularly, and as set forth initially in Parent application Serial No. 907,747, and the above-referenced applications that have followed, monocytes/macrophages were found to display high affinity surface binding activity specific for AGE moieties, independent of the protein which was AGE-modified. This macrophage AGE-receptor was shown to differ from other known scavenger receptors on these cells. In addition, an endogenous means for the jLn vivo elimination or removal of advanced glycosylation endproducts was set forth, and corresponding diagnostic applications involving the receptors and including a specific receptor assay were also proposed.

Following this determination, the applicants herein have sought to further investigate the identity and role of advanced glycosylation endproduct receptors and possible binding partners, and any consequent diagnostic and therapeutic implications of these investigations, and it is toward this end that the present invention is directed.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, receptors are disclosed that may be derived from either rat liver membranes, or mesangial cells and that recognize and bind advanced glycosylation endproducts. The receptors comprise at least one protein

and possess at least one of the following characteristics:

A. they recognize and bind with at least one ligand selected from AGE-BSA, AGE-RNase, AGE-Collagen I, and mixtures;

B. they do not recognize and bind with one or more of the ligands FFI-BSA, unmodified BSA, RNase, Collagen I, formaldehyde-treated BSA, glucosamide-BSA, and acetyl LDL-BSA in a solid phase ligand blotting assay; and C. they comprise at least one protein selected from the group consisting of a protein having a molecular mass of about 90 kD, a protein having a molecular mass of about 60 kD, a protein having a molecular mass of about 50 kD, a protein having a molecular mass of about 40 kD, and a protein having a molecular mass ranging from about 30 to about 35 kD, as determined by migration on SDS- PAGE.

In a further embodiment of the present invention, the receptors are derived from rat liver membrane cells and possess the following characteristics:

A. they recognize and bind with the ligands AGE- RNase and AGE-Collagen I;

B. they do not recognize and bind with the ligands FFI-BSA, formaldehyde-treated BSA, glucosamide-BSA, and acetyl LDL-BSA in a solid phase ligand blotting assay; and

C. they comprise at least two proteins, the first of said proteins having a molecular mass of about 90 kD and the second of said proteins having a molecular mass of about 60 kD as determined by their migration on SDS- PAGE.

In a still further embodiment of the present invention, the receptors are derived from mesangial cells and possess the following characteristics:

A. they recognize and bind with the ligands AGE- BSA, AGE-RNAse and AGE-collagen I in a saturable fashion, having a binding affinity of 2.0 ± 0.4 x 10 ⁶ M ^"1 (500nM) ;

B. they recognize and bind to AGE-BSA which has been reduced with NaBH ₄ ;

C. they do not recognize and bind with the ligand FFI-BSA, unmodified BSA, RNase or collagen I in a solid phase ligand blotting assay; and

D. they comprise at least one protein selected from the group consisting of a first protein having a molecular mass of about 50 kD, a second protein having a molecular mass of about 40 kD, and a third protein having a molecular mass of about 30-35 kD, as determined by their migration on SDS-PAGE.

More particularly, the mesangial cell-derived receptors may comprise a substantially purified receptor reactive with advanced glycosylation endproducts, characterized as follows:

(A) it contains at least one proteins selected from the group consisting of a protein having a molecular mass of about

50 kD, a protein having a molecular mass of about 40 kD and a protein having a molecular mass of about 30 - 35 kD;

(B) it is derived from mammalian mesangial cells;

(C) it is reactive with AGE-BSA, AGE-RNase, AGE-collagen I and AGE-BSA reduced with NaBH ₄ , having a binding affinity of

2.0 ± 0.4 X 10 ^*6 M ^*1 (kD = 500 nM) ;

(D) it is non-reactive with BSA, collagen I, RNase or chemically synthesized FFI-BSA; and

(E) it is present on mesangial cell membranes prior to purification in an amount sufficient to bind about 3.0 + 0.25

X 10 ⁵ AGE-modified protein molecules per mesangial cell.

The individual proteins listed above have certain common characteristics, to the extent that the proteins derived from rat liver cells are also expressed on both rat monocytes and macrophages, and both proteins copurify from elutions based,

respectively, on an AGE ligand affinity column, an anion exchange column, and a hydroxylapatite column. These proteins also have specific characteristics that distinguish them from each other, in that, when the proteins are immobilized on nitrocellulose in a solid phase assay such as that disclosed herein, the 90 kD protein does not bind to AGE-modified ligands while the 60 kD protein does.

A similar comparison of like properties and distinctions can be drawn with respect to the proteins that may comprise the receptor derived from mesangial cells. The preferred cells in this instance having receptors which are useful herein comprise mammalian mesangial cells; the preferred cellular components comprise cell membranes, and the cell proteins are derived from cell membranes and are selected from the group consisting of a 50 kD protein derived from MC membranes, a 40 kD protein derived from MC membranes, and a 30-35 kD protein derived from MC membranes, as well as mixtures thereof, having the reactivity which is described herein.

Further, with respect to the liver-derived receptors, the NH ₂ - terminal partial amino acid sequences of each of the proteins have been prepared and confirm that each protein is distinguishable from the other as well as to other known protein fractions as to sequence homology. The NH ₂ -terminal partial amino acid sequence for the 90 kD protein is presented below in FIGURE 11 and in Sequence ID No. 1, or SEQ ID NO:l, and comprises a single chain of 13 amino acids including two unidentified residues. The partial amino acid sequence depicted in SEQ ID N0:1 is reproduced below, with X representing the unidentified residues.

X Glu Val Lys Leu Pro Asp Met Val Ser Leu X Asp 1 5 10

The NH ₂ -terminal partial amino acid sequence for the 60 kD protein is presented in FIGURE 12 and in Sequence ID No. 2, or SEQ ID NO:2, and comprises a single chain of 22 amino acids

including two unidentified residues. The partial amino acid sequence depicted in SEQ ID NO:2 is reproduced below, with X representing the unidentified residues.

X Gly Pro Arg Thr Leu Val Leu Leu Asp Asn Leu Asn Val Arg 1 5 10

Arg Asp Thr His X Leu Phe Phe 15 20

The partial DNA sequence corresponding to the partial amino acid sequences of the proteins of the present invention or a portion thereof, or a degenerate variant of such partial DNA sequence, may be prepared as a probe to screen for complementary sequences and genomic clones in the same or alternate species, such as humans. The present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for clones that may corrrespond to genes expressing the respective proteins. For example, the probes may be prepared with a variety of known vectors. The present invention also includes the preparation of plasmids including such vectors.

The present invention also includes full proteins having the activities noted herein, and that display the partial amino acid seqences set forth and described above and with respect to SEQ ID NO:l and SEQ ID NO:2.

In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the 60kD and the 90kD protein, and more particularly, the complete DNA sequences determined from the partial sequences set forth above and in SEQ ID N0:1 and SEQ ID NO:2.

Likewise, the receptors and/or the proteins may be prepared alone or in operative association with another molecule or pharmaceutical agent in a form suitable for administration for either diagnostic or therapeutic purposes. The invention therefore extends to both diagnostic and pharmaceutical compositions including the receptors and/or the proteins, in combination with other diagnostic reagents in the former instance, and in combination with pharmaceutically acceptable carriers, and possibly, other therapeutic agents where coadministration is deemed appropriate or desirable.

Accordingly, while the exact role that the present receptors and the proteins play in the recognition and removal of AGEs and in tissue remodeling is as yet largely undefined, its participation in the elicitation of certain of these activities may be strongly inferred. The receptors and the proteins are therefore believed to possess significant diagnostic and therapeutic capabilities in connection with conditions involving the presence and activity of advanced glycosylation endproducts.

The receptors and the proteins may be prepared by isolation and purification from cells known to bear or produce the receptor and/or its protein, such as rat liver cells, monocytes and peritoneal acrophage. The cells or active fragments likely to participate in receptor protein synthesis or to have receptor protein associated therewith may be subjected to a series of known isolation techniques, such as for example elution of detergent-solubilized rat liver membrane proteins from an AGE-protein affinity matrix, whereupon the present receptor and/or the proteins may be recovered. Naturally, alternate procedures for preparation of the receptor and/or the proteins are contemplated and the invention is not limited to the procedures set forth herein.

The present invention also extends to antibodies including polyclonal and monoclonal antibodies, to the receptor and the

proteins that would find use in a variety of diagnostic and therapeutic applications. For example, the antibodies could be used to screen expression libraries to obtain the gene that encodes either the receptor or the proteins. Further, those antibodies that neutralize receptor protein activity could initially be employed in intact animals to better elucidate the biological role that the receptor and/or proteins plays. Such antibodies could also participate in drug screening assays to identify alternative drugs or other agents that may exhibit the same activity as the receptor proteins.

Possible therapeutic applications of the receptor and/or the proteins would include administration in instances where it is desirable to. stimulate the removal of advanced glycosylation endproducts and to correspondingly aid in the treatment of ailments where excess concentrations of AGEs may cause or exacerbate other dysfunctions or pathologies, such as diabetes.

The present invention also includes various diagnostic and therapeutic utilities predicated on the structure and activities of the receptor and/or the proteins. Diagnostic utilities include assays such as immunoassays with labeled quantities of the receptor proteins, antibodies, ligands and. binding partners thereto, receptor assays, and a drug screening assay to evaluate new drugs by their ability to promote or inhibit receptor protein production or activity, as desired. The above assays could be used to detect the presence and activity of the receptor proteins or of invasive stimuli, pathology or injury the presence or absence of which would affect receptor protein production or activity.

The present invention also extends to therapeutic methods and corresponding pharmaceutical compositions based upon the receptor proteins, and materials having the same or an antagonistic activity thereto. Therapeutic methods would be based on the promotion of the activities of the receptor

proteins and would extend to the treatment of diseases or dysfunctions attributable to the absence of receptor protein activity, and the concomitant excess in concentrations of AGEs in the host or patient such as for example, diabetic neuropathy, renal failure, atherosclerosis, stroke, cataracts, diabetic retinopathy, and the like. This method could be effected with the receptor, its proteins, their agonists or like drugs, or materials having a promotional effect on the production of the receptor proteins .in vivo.

Therapeutic compositions comprising effective amounts of the receptor, the receptor proteins, their agonists, antibodies, antagonists, or like drugs, etc. , and pharmaceutically acceptable carriers are also contemplated. Such compositions could be prepared for a variety of administrative protocols, including where appropriate, oral and parenteral administration. Exact dosage and dosing schedule would be determined by the skilled physician.

Diagnostic applications generally extend to a method for the measurement of protein aging both in plants and in animals, by assaying the presence, amount, location and effect of such advanced glycosylation endproducts. Assays of plant matter and animal food samples will be able for example, to assess food spoilage and the degradation of other protein material of interest so affected, while the assays of animals, including body fluids such as blood, plasma and urine, tissue samples, and biomolecules such as DNA, that are capable of undergoing advanced glycosylation, will assist in the detection of pathology or other systemic dysfunction.

Specifically, the methods comprise the performance of several competitive assay protocols, involving the analyte, a ligand and one or more binding partners to the advanced glycosylation endproducts of interest, where the binding partners are selected from the present receptor and/or the proteins. The binding partners may be generally selected from the group

consisting of rat liver cells as well as monocytes and macrophage having the present receptor and/or the proteins, cell components such as rat liver membranes, and the particular cell proteins set forth herein. The cell proteins are selected from the group consisting of the 90 kD protein derived from rat liver membranes, the 60 kD protein derived from rat liver membranes, and mixtures thereof.

The ligands useful in the present invention are generally AGE derivatives that bind to AGE binding partners. These ligands may be detected either singly and directly, or in combination with a second detecting partner such as avidin. Suitable ligands are selected from the reaction products of sugars such as glucose and glucose-6-phosphate with peptides, proteins and other bioche icals such as BSA, avidin, biotin, and enzymes such as alkaline phosphatase. Other suitable ligands may include synthetic AGEs or the reaction of the sugars directly with carriers capable of undergoing advanced glycosylation. Carriers not so capable may have a synthetic AGE coupled to them. Suitable carriers may comprise a material selected from carbohydrates, proteins, synthetic polypeptides, lipids, bio¬ compatible natural and synthetic resins, antigens, and mixtures thereof.

For example, standard assays based on either cell components or the cell proteins themselves and employing extract formats may be used. Each assay is capable of being based on enzyme linked and/or radiolabeled AGEs and their binding partners, including the AGE receptors disclosed herein. The broad format of assay protocols possible with the present invention extends to assays wherein no label is needed for AGE detection. For example, one of the formats contemplates the use of a bound protein-specific AGE receptor. In such instance, the analyte suspected of containing the advanced glycosylation endproducts under examination would need only to be added to the receptor, and the bound analyte could then be

easily detected by a change in the property of the binding partner, such as by changes in the receptor.

The assays of the invention may follow formats wherein either the ligand or the binding partner, be it a receptor or an antibody, are bound. Likewise, the assays include the use of labels which may be selected from radioactive elements, enzymes and chemicals that fluoresce.

Accordingly, it is a principal object of the present inventio to provide receptors for advanced glycosylation endproducts including receptor proteins in purified form.

It is a further object of the present invention to provide probes which facilitate screening of cDNA and genomic libraries in order to clone the animal and human genes encoding the receptor and/or the proteins.

It is a still further object of the present invention to provide the complete nucleic acid and corresponding amino acid sequences in both animals and humans for the receptor and/or the proteins.

It is a still further object of the present invention to provide agonists, antibodies, antagonists, and analogs thereof to the receptor and/or the proteins as aforesaid, compositions including pharmaceutical compositions containing them and methods for their discovery and preparation.

It is a still further object of the present invention to provide promoters of the synthesis of the receptor and/or the proteins as aforesaid, and methods for their preparation.

It is a further object of the present invention to provide a method for detecting the presence and amount of the receptor and/or the proteins and/or advanced glycosylation endproducts in mammals in which invasive, spontaneous, or idiopathic

pathological states related to excessive concentrations of advanced glycosylation endproducts are suspected to be present.

It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially effective in mimicking the activity of the receptor and/or the proteins in mammals.

It is a still further object of the present invention to provide a method for the treatment of mammals to modulate the amount or activity of the receptor and/or the proteins, so as to control the consequences of such presence or activity.

It is a still further object of the present invention to provide a method for the treatment of mammals to promote the amount or activity of the receptor and/or the proteins, so as to treat or avert the adverse consequences of excessive concentrations of advanced glycosylation endproducts regardless of origin.

It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the receptor and/or the proteins or their binding partner(s), or upon agents or drugs that control the production and/or activities of the receptor and/or the proteins.

It is a yet further object of the present invention to provide an assay for the measurement of advanced glycosylation endproducts that is capable of a broad range alternative protocols in accordance with the method as aforesaid.

It is a yet further object of the present invention to provide an assay as aforesaid that is capable of performance without

radioactive labels and that may be performed in automated fashion.

Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing description which proceeds with reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 is a graph depicting the relative binding and uptake of red blood cells modified with various agents, and illustrating a primary aspect of the present invention.

FIGURE 2 is a graph illustrating an assay in accordance with the present invention by the competitive inhibition in red blood cell binding caused by the introduction into a sample of an agent capable of stimulating red blood cells to increase their activity of recognition and removal of advanced glycosylation endproducts.

FIGURE 3 is a bar graph illustrating the comparative uptake and degradation of advanced glycosylation endproducts by mouse macrophages exposed to various stimulator compounds.

FIGURE 4 is a bar graph illustrating data similar to that set forth in FIGURE 3, with respect to one day old human monocytes.

FIGURE 5(A) - In vivo tissue distribution of ¹²⁵ I-AGE-RSA.

Fifty μg of either ^I25 I-AGE-rat serum albumin (AGE-RSA) (filled and striped bars) or ^I25 I-native RSA (shaded bars) were administered i.v. to normal rats. At 10 min the animals were sacrificed, and the organs and tissues were removed and counted for radioactivity. Whole organ counts were corrected for blood-associated counts as described in Methods.

FIGURE 5(B) - Specific competition of ¹²⁵ I-AGE-RSA uptake in rat liver, spleen, lung and kidney. Immediately prior to receiving radio-labeled ligand, rats were injected with excess non-labeled AGE-RSA (5 mg, i.v.). At the indicated time intervals, incorporated radioactivity was determined in blood and tissues, and compared according to a Tissue-to-Blood Isotope Ratio (TBIR) formula as described in text. Closed and striped bars: ¹²⁵ I-AGE-RSA (50 μg) alone. Open bars: ^1S I-AGE- RSA (50 μg) in the presence of excess non-labeled AGE-RSA. Data are expressed as mean ± SEM of three independent measurements performed in five animals per group.

FIGURE 6 depicts several graphs of an AGE-binding activity assay: (A) Relationship of liver membrane protein concentration to AGE-licrand binding. Aliquots (1-50 μg) of a detergent-solubilized liver membrane protein preparation were immobilized on nitrocellulose filters, incubated with blocking buffer, and then probed for AGE-protein binding activity with ¹²⁵ I-AGE-BSA in the presence or absence of excess non-labeled AGE-BSA. After washing, the blots were counted for ¹²⁵ I. Closed circles: ¹²⁵ I-AGE-BSA alone (total binding) . Open circles: ¹²⁵ I-AGE-BSA plus 100-fold excess non-labeled AGE-BSA (non-specific binding) . Data points represent duplicate blots. (B) Saturability of ^{l 5} I-AGE-BSA binding. A fixed amount of solubilized liver membrane proteins (8 μg) was immobilized on duplicate nitrocellulose filters which were probed with increasing concentrations of ^l25 I-AGE-BSA (10-100 nM, s.a. = 1.5 X 10 ⁵ cpm/ng) in the presence (non-specific) or absence (total binding) of 200-fold excess non-labeled AGE-BSA. After washing, the nitrocellulose filters were counted for retained ^l25 I. Specific binding was determined by subtracting non-specific binding from total binding. (C) Scatchard analysis of specific binding data (B = pmoles/8 μg membrane protein, F = μM) . (D) Effects of differently modified protein ligands on ¹²⁵ I-AGE- BSA binding. Filter blots of solubilized liver membranes prepared as above were probed with ^I25 I-AGE-BSA (50 nM) alone.

or in the presence of 150-fold excess of various non-labeled competitors: AGE-albumin (AGE-BSA) , AGE-ribonuclease (AGE- RNAse) , AGE-collagen I, FFI-BSA, formaldehyde-treated albumin (f-alb) , glucosamide BSA, acetyl-LDL (act-LDL) . Data are expressed as the amount of ¹²⁵ I-AGE-BSA (cpm X 10 ^-3 ) retained on duplicate blots.

FIGURE 7 shows the purification of rat liver AGE-binding proteins. Detergent-solubilized membrane proteins were fractionated by successive PEI-cellulose, DEAE-cellulose, and BSA-Sepharose 4B column chromatography, as described in Methods. The flow-through from the BSA-Sepharose column was then applied to an AGE-BSA-Sepharose 4B column. This column was washed and bound proteins were eluted by the addition of high salt buffer. Each fraction was concentrated and analyzed for AGE-binding activity using the binding assay described in Figure 6. Open circles: total binding activity; closed circles: non-specific activity. Inset: SDS-PAGE analysis of fraction #8 (mercaptoethanol reduced) , and stained with Coommassie Blue.

FIGURE 8 - (A) Ligand blot analysis of rat liver membrane proteins. Aliquots (15 μg each) of detergent-solubilized membrane proteins were electrophoresed through 8-16% acrylamide gradient gel under non-reducing conditions and eletro-transferred onto nitrocellulose filters. Using ¹²⁵ I-AGE- BSA (50 nM, 8.0 X 10 ⁵ cpm/ng) as probe, specific binding was determined in the presence of 0, 25, or 150-fold excess non- labeled AGE-BSA using autoradiographic detection. Migration of molecular weight standards is shown at left. (B)

Aliquots of solubilized membrane proteins (mercaptoethanol reduced) , were electrophoresed through an 8-16% gradient gel and electro-transferred onto nitrocellulose filters. After washing and blocking with excess BSA, the blots were probed with purified IgG fractions of either anti-p60 or anti-p90 chicken antisera or the corresponding preimmune sera, and then exposed to goat anti-chicken antibody conjugated to alkaline

phosphatase and reacted for phosphatase-dependent color development. Lane 1, preimmune IgG; Lane 2, anti-p60 IgG; Lane 3, preimmune IgG, Lane 4, anti-p90 IgG. Results are representative of three independent experiments.

FIGURE 9 - Demonstration by flow cytometry of expression of p60 and p90 AGE-binding proteins rat monocytes and macrophages. Peripheral blood monocytes (A-D) and peritoneal resident macrophages (E and F) were treated with biotinylated anti-p60 alone (A and E) , biotinylated anti-p90 alone (B and F) , biotinylated anti-p60 + 20-fold excess unconjugated anti- p90 (C) , biotinylated anti-p90 + 20-fold excess unconjugated anti-p60 (D) , followed by FITC-avidin and analyzed by FACSCAN. Arrow 1: Fluorescence of cells treated with FITC-avidin in the absence of either anti-p60 or anti-p90 (dotted line) . Arrow 2: Fluorescence of cells treated with FITC-avidin subsequent to treatment with biotinylated chicken IgG (isotypic control) . Arrow 3: Fluorescence of cells treated with FITC-avidin subsequent to treatment of cells with biotinylated anti-p60 (A,C,E) and anti-p90 (B,D,F). Note the lack of competition between a 20-fold excess of unconjugated anti-p90 for the binding of anti-p60 to the monocytes (panel A vs C) , and the lack of competition between a 20-fold excess of unconjugated anti-p60 for the binding of anti-p90 to the monocyte cell surface (panel B vs D) . All antibodies were used at a final concentration of 5 μg/ml, unless otherwise indicated.

FIGURE 10 - Inhibition of ^l25 I-AGE-BSA binding (A) and ¹²⁵ I-FFI- BSA binding (B) on rat macrophage cell surface by anti-p60 and anti-p90 antibodies. Rat peritoneal resident macrophages were collected by peritoneal lavage and purified, then incubated with the indicated radio-labeled ligand in the presence or absence of a 10-fold excess of non-labeled ligand or in the presence of antibodies p60 or p90-specific at the indicated dilutions. Both antibodies were used alone (undiluted: 2 μg/200 μl) or in combination (at 1:10 dilution). Data are expressed as % of maximal control binding (defined as the

amount of ^I25 I-ligand bound to the cell surface in the presence of 10% FBS) and represent the mean of duplicate experiments.

FIGURE 11 depicts the NH ₂ -terminal partial amino acid sequence prepared by blotting a quantity of gel-purified rat liver membrane protein having a molecular mass of about 90 kD (p90) onto Immobilon membranes. The amino acids are numbered from 1 to 13. This sequence is identically depicted in the SEQUENCE LISTING presented later on herein, in accordance with 37 C.F.R. 1.821-825, enacted October 1, 1990, and is cumulatively and alternately referred to as SEQ ID NO:l.

FIGURE 12 depicts the NH ₂ -terminal partial amino acid sequence prepared by blotting a quantity of gel-purified rat liver membrane protein having a molecular mass of about 60 kD (p60) onto Immobilon membranes. The amino acids are numbered from 1 to 22. This sequence is identically depicted in the SEQUENCE LISTING presented later on herein, in accordance with 37 C.F.R. 1.821-825, enacted October 1, 1990, and is cumulatively and alternately referred to as SEQ ID NO:2.

FIGURE 13 - Binding of '"i-AGE-BSA to human fA ^* ι and rat fBI MC membranes. 10 μg of solubilized membrane protein from human and rat mesangial cells were dot-blotted onto nitrocellulose filters, which were then incubated with various concentrations of ^I5 I-AGE-BSA in the presence and absence of 100-fold excess unlabeled AGE-BSA. Specific binding was obtained by subtracting the nonspecific binding from the total binding. The inset shows a Scatchard Plot for the specific binding (B = pmoles/ng membrane protein, F = nM) .

FIGURE 14 -Competitive inhibition of ¹²⁵ I-AGE-BSA binding to human (A) and rat (B) MC membranes. 10 μg of solubilized membrane protein from human and rat MCs were dot-blotted onto nitrocellulose filters. The filters were incubated with 50 nM ^,25 I-AGE-BSA for 2 hours at 4°C. Competition experiments were performed in parallel experiments in which the radioligand was

incubated with 100-molar excess of an unlabeled protein. Data shown are the average of duplicate determinations, and are expressed as the % maximal binding. Maximal binding was defined as the amount of ^,25 I-AGE-BSA bound in the presence of 100-molar excess cold BSA. Competitors used: (a) BSA, (b)

AGE-BSA, (c) NaBH ₄ -reduced AGE-BSA, (d) FFI-BSA, (e) AGE-RNAse, (f) RNAse, (g) Collagen I, (h) AGE-collagen I.

FIGURE 15 - A) Uptake and degradation of ¹²⁵ I -AGE-BSA by rat MCs. MCs were incubated with various concentrations of

¹²⁵ I-AGE-BSA for 4 hours at 37°C. The amount of cell-associate ^12S I-AGE-BSA (uptake) , and the amount of trichloroacetic acid-soluble counts in the medium (degradation) were determined in triplicate wells. B) Accumulation of ¹²⁵ I-AGE-BS versus time. MCs in each well were incubated with 20 μg of ¹²⁵ I-AGE-BSA at 37°C and specific cell-associated radioactivity was determined at various time intervals. Cellular accumulation of radioactivity is expressed as the % of the maximal accumulation of ^*25 I-AGE-BSA.

FIGURE 16 - Ligand blot analysis of enriched human MC membranes. 10 μg of solubilized membrane protein were electrophoresed on a nonreducing SDS/polyacrylamide gel (10%) . The proteins on the gel were electroblotted onto nitrocellulose membrane and probed with ^I25 I-AGE-BSA in the presence of 100-fold excess of either BSA (lane a) or AGE-BSA (lane b) . The analysis presented is one of four identical experiments.

FIGURE 17 - Effects of AGE-matrices on r ³ H1thymidine incorporation by MCs. Rat MCs were plated onto various matrices (10 μg/ml) , as described: (a) Fibronectin, (b) AGE- Fibronectin, (c) Collagen I, (d) AGE-Collagen I, (e) Laminin, (f) AGE-Laminin. The results are expressed as the means ± SEM of 6 experiments and are expressed as the % of [ ³ H] hymidine incorporated relative to the control value, with control

representing [ ³ H]thymidine incorporated by cells plated on plastic.

FIGURE 18 - Effect of AGE- atrices on fibronectin synthesis by MCs. Human MCs were plated onto either unmodified or

AGE-modified matrices and labeled with 35S-methionine and cysteine, as described. The amount of fibronectin released into the medium (A) , and incorporated into the matrices (B) , was determined by i munoprecipitation. The fibronectin bands on the gel were excised and counted for radioactivity. The values shown are expressed as the % increase in fibronectin produced by cells plated on the AGE-matrices relative to that produced by cells plated on control unmodified matrices. The values show cpm/well and represent the means ± SEM from 4 experiments. a) Fibronectin, b) Polylysine, c) Collagen I.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g.. Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual" (1982) ; "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985) ; "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" (B.D. Hames & S.J. Higgins eds. 1985); "Transcription And Translation" (B.D. Hames & S.J. Higgins eds. 1984); "Animal Cell Culture" (R.I. Freshney ed. 1986) ; "Immobilized Cells And Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide To Molecular Cloning" (1984) .

Therefore if appearing herein, the following terms shall have the definitions set out below.

The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D"

isomeric form can be substituted for any L-amino acid residue,, as long as the desired fuctional property of immunoglobulin- binding is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J. Biol. Chem. , 243:3552-59 (1969) , abbreviations for amino acid residues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE

It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right

orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.

As used throughout the present application, the terms "receptor" and "receptor protein(s)" include both the singular and plural and contemplates the existence of a single receptor structure comprised as all or part thereof, of the individual proteins defined herein, or a plurality of receptor structures respectively constituted in whole or in part by individual of said proteins. This definition is therefore to be explicitly distinguished from the definition that may be inferred from the term as it appears in the manuscript by Yang et al. from which the present application is drawn in part.

An "antibody" is any immunoglobulin, including antibodies and fragments thereof, such as Fab, F(ab') ₂ or dAb, that binds a specific epitope. The term encompasses, inter alia, polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567. An antibody preparation is reactive for a particular antigen when at least a portion of the individual immunoglobulin molecules in the preparation recognize (i.e., bind to) the antigen. An antibody preparation is non-reactive for an antigen when binding of the individual immunoglobulin molecules in the preparation to tlie antigen is not detectable by commonly used methods.

More particularly, the present invention relates to receptors including proteins as defined herein that are derived from either rat liver membranes, as well as monocytes and peritoneal macrophage, or from mesangial cells, and that recognize and bind advanced glycosylation endproducts. The

receptors possess at least one of the following characteristics:

A. They recognize and bind with at least one ligand selected from AGE-BSA, AGE-RNase, AGE-Collagen I, and mixtures;

B. They do not recognize and bind with one or more of the ligands FFI-BSA, unmodified BSA, RNase, Collagen I, formaldehyde-treated BSA, glucosamide-BSA, and acetyl LDL-BSA in a solid phase ligand blotting assay; and C. They comprise at least one protein selected from the group consisting of a protein having a molecular mass of about 90 kD, a protein having a molecular mass of about 60 kD, a protein having a molecular mass of about 50 kD, a protein having a molecular mass of about 40 kD, and a protein having a molecular mass ranging from about 30 to about 35 kD, as determined by migration on SDS-PAGE.

In a further embodiment of the present invention, the receptors are derived from rat liver membrane cells and possess the following characteristics:

A. they recognize and bind with the ligands AGE-RNase and AGE-Collagen I;

B. they do not recognize and bind with the ligands FFI- BSA, formaldehyde-treated BSA, glucosamide-BSA, and acetyl

LDL-BSA in a solid phase ligand blotting assay; and

C. they comprise at least two proteins, the first of said proteins having a molecular mass of about 90 kD and the second of said proteins having a molecular mass of about 60 kD as determined by their migration on SDS-PAGE.

In a still further embodiment of the present invention, the receptors are derived from mesangial cells and possess the following characteristics: A. they recognize and bind with the ligands AGE-BSA, AGE-RNAse and AGE-collagen I in a saturable fashion, having a binding affinity of 2.0 ± 0.4 x 10 ⁶ M ^*1 (500nM) ;

B. they recognize and bind to AGE-BSA which has been reduced with NaBH ₄ ;

C. they do not recognize and bind with the ligand FFI- BSA, unmodified BSA, RNAse or collagen I in a solid phase ligand blotting assay; and

D. they comprise at least one protein selected from the group consisting of a first protein having a molecular mass of about 50kD, a second protein having a molecular mass of about

40kD, and a third protein having a molecular mass of about 30- 35 kD, as determined by their migration on SDS-PAGE.

The individual proteins listed above have certain common characteristics, to the extent that the proteins derived from rat liver cells are also expressed on both rat monocytes and macrophages, and both proteins copurify from elutions based, respectively, on an AGE ligand affinity column, an anion exchange column, and a hydroxylapatite column. These proteins also have specific characteristics that distinguish them from each other, in that, when the proteins are immobilized on nitrocellulose in a solid phase assay such as that disclosed herein, the 90 kD protein does not bind to AGE-modified ligands while the 60 kD protein does.

As stated earlier, a similar comparison of like properties and distinctions can be drawn with respect to the proteins that may comprise the receptor derived from mesangial cells.

As set forth earlier, the NH ₂ -terminal partial amino acid sequences of each of the proteins of the liver-derived receptors have been prepared and confirm that each protein bears no homology with the other as well as with other known protein fractions. The NH ₂ -terminal partial amino acid sequence for the 90 kD protein is presented below and in SEQ ID NO:l, and comprises a single chain of 13 amino acids including two unidentified residues represented by "X".

X Glu Val Lys Leu Pro Asp Met Val Ser Leu X Asp 1 5 10

The NH ₂ -terminal partial amino acid sequence for the 60 kD protein is presented below in SEQ ID NO:2, and comprises a single chain of 22 amino acids including two unidentified residues, likewise represented by "X".

X Gly Pro Arg Thr Leu Val Leu Leu Asp Asn Leu Asn Val Arg 1 5 10

Arg Asp Thr His X Leu Phe Phe 15 20

As stated earlier, the partial DNA sequence corresponding to the partial amino acid sequences of the proteins of the present invention or a portion thereof, may be prepared as a probe to screen for complementary sequences and genomic clones in the same or alternate species, such as humans. The present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for clones that may corrrespond to genes expressing the respective proteins. For example, the probes may be prepared with a variety of known vectors, such as phage λ vectors. The present invention also includes the preparation of plasmids including such vectors.

The present invention also includes full proteins having the activities noted herein, and that display the partial amino acid seqences set forth and described above and with respect to SEQ ID NO:l and SEQ ID NO:2.

With respect to the mesangial cell-derived receptors, mesangial cells are contained in the mammalian kidneys and function in conjunction with the glomeruli to regulate the glomerular filtration rate, thus affecting glomerular flow.

Increases in the mesangial matrix have been shown to decrease the filtering surface of the glo erulus, and thus impinge upon the glomerular capillary vasculature. This is due to the accumulation of normal matrix proteins, e.g., collagen type

IV, type V, laminin and fibronectin.

Mesangial cells have been evaluated as described herein, and found to contain AGE receptors. The specific findings that follow derive from the experiments the procedures of which are set forth in detail in Example 4, presented below. When AGE- modified proteins accumulate in the mesangial matrix and bind to these receptors, MC proliferation, synthesis, metabolism and physiology are modified. For example, the proliferation of MCs is reduced when AGEs have reacted with MC receptors as compared to MCs which have not been exposed to AGEs.

Additionally, when AGE-modified proteins are bound to MCs, an increase in fibronectin production is observed, which in turn causes an adverse build-up of the mesangial matrix.

The MC receptors which recognize AGEs are present on the cell membranes, and demonstrate binding to AGE-modified proteins in a saturable fashion with a binding affinity of about 2.0 ± 04 x 10 ⁶ M ^" ' (kD = 500 nM) . This binding is specific for AGE- modified proteins; non-AGE modified proteins do not compete for receptor recognition in binding assays.

The MC receptor complex for AGEs is comprised of three distinct proteins, 50kD, 40kD and 30-35 kD. This was demonstrated using a ligand blotting assay of MC membrane extracts, the procedure of which is described below. The results are shown in FIGURE 16.

The MC AGE receptor was further characterized with respect to its binding affinity, using a series of assays. It was determined that the MC membrane AGE receptors bind AGEs in a saturable fashion. (See FIGURE 13.) When MC membrane extracts were exposed to increasing levels of AGEs, specific binding plateaued, even as AGE levels were increased. Half- maximal binding occurred at about a 150nM concentration of AGE-BSA. This was consistent between rat and human MC membrane extracts. The number of AGE molecules bound per cell

was in the range of 3.0 ± 0.25 x 10 ⁵ molecules per cell. The binding affinity constant was 2.0 ± 0.40 x 10 ⁶ M" ¹ (kD - 500nM) .

Similar results were observed when whole MCs were assayed (data not shown) .

The MC receptor binding affinity was further evaluated, and it was determined that the MC receptor for AGE- odified proteins is reactive with AGE-modified proteins and non- reactive with proteins in unmodified form. These conclusions were drawn based upon competitive binding assays run wherein the MC receptor for AGEs is dot-blotted onto a nitrocellulose filter, blocked with BSA, quantitated with labelled ligand and then competitive assayed with labelled ligand and the protein to be evaluated. Competition was evaluated based upon the level of reactivity, compared to that which was present when no competing protein was included.

Specific binding to the receptors was defined as the difference between total binding (radioligand incubated with membrane protein alone) and non-specific binding (cell incubated with radiolabelled ligand plus 100 fold excess of unlabelled ligand) .

Scatchard analysis of the data was performed to determine the binding affinity constant and the receptor number. See Scatchard, G. Ann. N.Y. Acad. Sci. (1949) 5_1: 660-72.

The competitive binding assays were run using rat and human.MC extracts, with the results shown in FIGURE 14. It was confirmed that AGE-modified proteins were binding to the receptors. Excess cold AGE-BSA (Fig. 14) competed with labelled AGE-BSA. The excess cold AGE-BSA competitively inhibited greater than 80% of ^l25 I-AGE-BSA binding to MC membrane extract, and other AGE-modified proteins, namely AGE- RNAse (FIGURE 14), and AGE-collagen I (FIGURE 14) competed effectively with ¹²⁵ I-AGE-BSA.

Unmodified BSA (FIGURE 14) did not compete, nor did excess unmodified RNAse or collagen I (FIGURE 14) .

The AGE-modified protein receptors were further characterized with respect to binding for AGE-BSA in reduced form. AGE-BSA was reduced with NaBH ₄ to glucitolysine and further evaluated. The reduced AGE-BSA effectively competed with radiolabelled AGE-BSA for binding to MC membrane extracts (FIGURE 14) .

The chemically synthesized model AGE-, FFI was assessed for its competitive ability to bind to MC AGE-receptors. The results are shown in Fig. 14. It did not compete with labelled AGE-BSA. The MC receptor does not recognize FFI-BSA.

The characteristics of the MC AGE-modified protein receptor were evaluated to determine the number of proteins involved and their molecular weight. It was determined that three distinct membrane proteins are present in the receptor complex, 50kD, 40kD and 30-35 Kd, using a ligand blot analysis.

The receptor proteins were evaluated using detergent extracts of MC membranes. FIGURE 16 shows three prominent AGE binding proteins. Binding of radiolabelled AGE to these membrane proteins was specific. Again, excess unlabelled AGE-BSA could inhibit labelled AGE-binding for both rat and human MCs, whereas BSA did not inhibit labelled AGE binding.

The effect of AGEs on mesangial cell metabolism was evaluated using a number of different comparisons. Typically, the analysis addressed a particular metabolic parameter in AGE- modified MCs, and compared it to MCs unexposed to AGEs. To evaluate the effect of AGEs on MC proliferation, MCs were incubated with and without AGE-modified proteins in the presence of labelled thymidine. (Thymidine is taken up during DNA synthesis) . The results are shown in FIGURE 17. AGE-

modified MCs showed a decrease in label uptake over MCs not exposed to AGEs.

MC proliferation was also evaluated in the presence of matrix proteins, fibronectin, collagen I and laminin, comparing MC growth on plates coated with these matrix proteins to that which occurs in the presence of these matrix proteins in AGE modified form. The results are shown in FIGURE 17. Labelled thymidine uptake was used as the measure of MC proliferation. When used in a concentration of 10 mg/ml, the uptake of labelled thymidine uptake was consistently reduced in the presence of AGE-modified matrix proteins, approximately to the level of incorporation in MCs plated on plastic.

In contrast cells grown on collagen I and fibronectin coated plates (non-AGE modified) showed enhanced thymidine uptake; cells grown on non-AGE modified laminin coated plates showed no stimulated uptake over control values.

The reduced thymidine uptake in the presence of AGE modified fibronectin, collagen I and laminin was confirmed with Brd U incorporation assays used to control for DNA synthesis.

The uptake and degradation of AGEs by MCs was also evaluated using the procedures of Vlassara, H. et al. Proc. Natl. Acad. Sci. USA (1985); 82:5588-92 (modified slightly). MC accumulation of labelled ligand (AGE-BSA) was assessed by incubating cells with various concentrations of labelled AGE- BSA in the presence or absence of a 100 fold excess of unlabelled AGE-BSA. The amount of cell-contained label was determined. This is generally indicative of AGE uptake by MCs. The results are shown in FIGURE 15. Uptake data is shown in FIGURE 15. MCs continued to accumulate AGEs beyond the level of AGE-binding to as high as l.l μM.

Since the level of AGE accumulation could include both bound AGEs and internalized AGEs, accumulation at 4°C was compared

to that at 37°C. (At 4°C AGEs bind to MCs, whereas at 37°C. AGEs are internalized by MCs.) The maximum accumulation of labelled AGE-BSA occurred within 2 hours of incubation at 37°C (FIGURE 15) . The amount of label which was cell associated at 37°C was 2-4 times higher than the amount of label bound to MCs at 4°C.

Degradation of AGEs was determined by measuring the amount of label present in the aspirated medium.

Concommitant with MC accumulation of labelled AGE-BSA, ligand degradation also increased, as measured by a steady increase in trichloro acetic acid (TCA) soluble radioactivity in the media. The increase in AGE-BSA degradation paralleled the increase in MC uptake of AGE-BSA. (FIGURE 15) .

The effect of AGEs on fibronectin production in MCs was also evaluated. Mesangial cells were grown on AGE-modified or unmodified collagen I, fibronectin and polylysine media. The amount of fibronectin released into the medium (or incorporated into the matrix) was determined by immunoprecipitation with IgG purified anti-human fibronectin antibodies. The results are shown in FIGURE 18, showing an increase in the synthesis of fibronectin in the presence of AGE- modified proteins.

The mesangial cells receptors for AGE- modified proteins can further be characterized by comparing said receptors to other known AGE- receptors, such as on macrophage and endothelial cells. AGEs have been shown to bind to specific receptors on murine and human monocyte/macrophages, as well as on bovine and human endothelial cells. Since AGEs form progressively over time, the function of AGE receptors is believed to include signalling cells to promote turnover of aging tissue. Under normal conditions, the removal of AGE- modified proteins occurs at a rate which keeps up with production, thereby preventing accumulation.

In diabetes, excessive AGE formation in the presence of elevated glucose levels results in a net increase of AGEs on most structural tisue proteins.

Macrophages are induced to release the cytokines catechin/tumor necrosis factor (TNF) , interleukin-1 (IL-1) , platelet derived growth factor (PDGF) ,__ and insulin-like growth factor (ILGF) . Excessive blood glucose levels may lead to an exaggerated response, which could contribute to complications, e.g., atherosclerotic plaque formation, mesangial expansion and the like.

Endothelial cell AGE receptors induce several changes in EC functionwhich are characteristic in diabetes, such as increased EC permeability and procoagulant properties.

The macrophage AGE receptors recognize the model AGE, FFI-BSA; in contrast, endothelial cell AGE receptors do not recognize the AGE- FFI-BSA.

The ability of MCs to slowly internalize and degrade AGE- modified proteins points to a contribguting role for MCs in the turnover and remodeling of senescent AGE- modified mesangial matrix proteins. The efficiencywith which MCs ingest and degrade AGE-BSA is approximately 10% of that which has been reported for the macrophage. While the AGE receptors on MCs, macrophage cells and endothelial cells all recognize AGEs to a degree, there are other differences. First, the Kds for the receptors are different. The Kd for macrophage and endothelial cell AGE receptors is about 70-100nM; the Kd for MC AGE receptors is about 500nM.

In addition, the efficiencies at which the AGE receptors from these three cell types internalize and degrade AGE- modified proteins are different. Macrophage AGE receptors ingest and degrade AGE-BSA at a much higher rate than do endothelial cells or MCs.

Lastly, macrophage AGE receptors recognize the specific adduct FFIO-BSA,whereas endothelial cell and MC AGE receptors do not. These differences in reactivity can be used to characterize the AGE receptor proteins derived from their respective cell types.

As stated earlier, the receptor and/or the proteins may be prepared by isolation and purification from cells known to bear or produce the receptor and/or the proteins, such as rat liver cells, monocytes and peritoneal macrophage. The cells or active fragments likely to participate in receptor protein synthesis or to have receptor protein associated therewith may be subjected to a series of known isolation techniques, such as for example elution of detergent-solubilized rat liver membrane proteins from an AGE-protein affinity matrix, whereupon the present receptor and/or the proteins may be recovered. A specific protocol is set forth by way of illustration in Example III, later on herein. The present invention naturally contemplates alternate means for preparation of the receptor and/or the proteins, including stimulation of receptor producer cells with promoters of receptor synthesis followed by the isolation and recovery of the receptor as indicated above, as well as chemical synthesis, and the invention is accordingly intended to cover such alternate preparations within its scope.

The present invention also extends to antibodies including polyclonal and monoclonal antibodies, to the receptor and the proteins that would find use in a variety of diagnostic and therapeutic applications. For example, the antibodies could be used to screen expression libraries to obtain the gene that encodes either the receptor or the proteins. Further, those antibodies that neutralize receptor protein activity could initially be employed in intact animals to better elucidate the biological role that the receptor and/or the proteins plays. Such antibodies could also participate in drug

screening assays to identify alternative drugs or other agents that may exhibit the same activity as the receptor proteins.

Both polyclonal and monoclonal antibodies to the receptor are contemplated, the latter capable of preparation by well known techniques such as the hybridoma technique, utilizing, for example, fused mouse spleen lymphocytes and myeloma cells. Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with

Epstein-Barr virus. Specific avian polyclonal antibodies were raised herein and are set forth in Example III. Naturally, these antibodies are merely illustrative of antibody preparations that may be made in accordance with the present invention.

As the receptor and the proteins appear to play a role in the recognition and removal of advanced glycosylation endproducts in vivo, the present invention contemplates both diagnostic and therapeutic applications for these agents. Accordingly, the receptor and/or the proteins may be prepared for use in a variety of diagnostic methods, set forth in detail hereinafter, and may be labeled or unlabeled as appropriate. Likewise, the receptor and/or the proteins may be prepared for administration in various scenarios for therapeutic purposes, in most instances to assist in reducing the concentration of AGEs in vivo.

The receptor and/or the proteins may be prepared in a therapeutically effective concentration as a pharmaceutical composition with a pharmaceutically acceptable carrier. Other compatible pharmaceutical agents may possibly be included, so that for example certain agents may be simultaneously coadministered. Also, the receptor and/or the proteins may be associated with or expressed by a compatible cellular colony, and the resulting cellular mass may then be treated as a therapeutic agent and administered to a patient in accordance

with a predetermined protocol. Numerous therapeutic formulations are possible and the present invention contemplates all such variations within its scope. A variety of administrative techniques may be utilized, among them topical applications as in ointments or on surgical and other topical appliances such as, surgical sponges, bandages, gauze pads, and the like. Also, such compositions may be administered by parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, catheterizations and the like.

Corresponding therapeutic utilities take advantage of the demonstrated activity of the present receptor and/or the proteins toward advanced glycosylation endproducts. Thus, to the extent that the in vivo recognition and removal of AGEs serves to treat ailments attributable to their presence in an excess concentration, the administration of the present receptor and/or the proteins comprises an effective therapeutic method. Such conditions as diabetic nephropathy, renal failure and the like may be treated and/or averted by the practice of the therapeutic methods of the present invention. Average quantities of the active agent may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian, with an exemplary dosage regimen extending to up to about 25 mg/kg/day.

The present invention also relates to a variety of diagnostic applications, including methods for the measurement of the presence and amount of advanced glycosylation endproducts in both plants and animals, including humans. The methods comprise assays involving in addition to the analyte, one or more binding partners of the advanced glycosylation endproducts, and one or more ligands.

Accordingly, the present assay method broadly comprises the steps of:

A. preparing at least one biological sample suspected of containing said advanced glycosylation endproducts;

B. preparing at least one corresponding binding partner directed to said samples, wherein said binding partner includes or is selected from the present receptor, the receptor proteins, and mixtures;

C. placing a detectable label on a material selected from the group consisting of said samples, a ligand to said binding partner and said binding partner;

D. placing the labeled material from Step C in contact with a material selected from the group consisting of the material from Step C that is not labeled; and

E. examining the resulting sample of Step D for the extent of binding of said labeled material to said unlabeled material.

Suitable analytes may be selected from plant matter, blood, plasma, urine, cerebrospinal fluid, lymphatic fluid, and tissue; and the compounds FFI and AFGP, individually and bound to carrier proteins such as the protein albumin. The analyte may also comprise a synthetically derived advanced glycosylation endproduct which is prepared, for example, by the reaction of a protein or other macromolecule with a sugar such as glucose, glucose-6-phosphate, or others. This reaction product could be used alone or could be combined with a carrier in the same fashion as the FFI-albumin complex.

The carrier may be selected from the group consisting of carbohydrates, proteins, synthetic polypeptides, lipids, bio¬ compatible natural and synthetic resins, antigens and mixtures thereof.

As stated earlier, the present invention seeks by means of the present receptor to diagnose both the degradative effects of advanced glycosylation of proteins in plants and the like, and the adverse effects of the buildup of advanced glycosylation

endproducts in animals. Such conditions as age- or diabetes- related hardening of the arteries, skin wrinkling, arterial blockage, and diabetic, retinal and renal damage in animals all result from the excessive buildup or trapping that occurs as advanced glycosylation endproducts increase in quantity. Therefore, the diagnostic method of the present invention seeks to avert pathologies caused at least in part by the accumulation of advanced glycosylation endproducts in the body by monitoring the amount and location of such AGEs.

Likewise, as advanced glycosylation endproducts may be measured by the extent that they bind to receptors on cells from a variety of sources, the assays of the present invention are particularly suited to design and performance around this activity. For example, in a typical competitive assay in accordance with the present invention, the present receptor or and/or the proteins and/or cellular material bearing the receptor may be combined with the analyte and the ligand and the binding activity of either or both the ligand or the analyte to the receptor may then be measured to determine the extent and presence of the advanced glycosylation endproduct of interest. In this way, the differences in affinity between the components of the assay serves to identify the presence and amount of the AGE.

The present invention also relates to a method for detecting the presence of stimulated, spontaneous, or idiopathic pathological states in mammals, by measuring the corresponding presence of advanced glycosylation endproducts. More particularly, the activity of AGEs may be followed directly by assay techniques such as those discussed herein, through the use of an appropriately labeled quantity of at least one of the binding partners to AGEs as set forth herein. Alternately, AGEs can be used to raise binding partners or antagonists that could in turn, be labeled and introduced into a medium to test for the presence and amount of AGEs therein.

and to thereby assess the state of the host from which the medium was drawn.

Thus, both AGE receptors and any binding partners thereto that may be prepared, are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, a receptor or other ligand to an AGE that may either be unlabeled or if labeled, then by either radioactive addition, reduction with sodium borohydride, or radioiodination.

In an immunoassay, a control quantity of a binding partner to advanced glycosylation endproducts may be prepared and optionally labeled, such as with an enzyme, a compound that fluoresces and/or a radioactive element, and may then be introduced into a tissue or fluid sample of a mammal believed to be undergoing invasion. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.

The presence of AGE activity in animals and plants can be ascertained in general by immunological procedures are which utilize either a binding partner to the advanced glycosylation endproduct or a ligand thereto, optionally labeled with a detectable label, and further optionally including an antibody Ab, labeled with a detectable label, an antibody Ab ₂ labeled with a detectable label, or a chemical conjugate with a binding partner to the advanced glycosylation endproduct labeled with a detectable label. The procedures may be summarized by the following equations wherein the asterisk indicates that the particle is labeled, and "BP" in this instance stands for all binding partners of advanced glycosylation endproduct(s) under examination:

A. BP* + Ab, = BP*Ab,

B . BP + Ab* = BPAb, *

C . BP + Ab, + Ab ₂ * = BPAb,Ab ₂ *

D. Carrier*BP + Ab, = Carrier*BPAb,

These general procedures and their application are all familiar to those skilled in the art and are presented herein as illustrative and not restrictive of procedures that may be utilized within the scope of the present invention. The "competitive" procedure, Procedure A, is described in U.S. Patent Nos. 3,654,090 and 3,850,752. Optional procedure C, the "sandwich" procedure, is described in U.S. Patent Nos. RE 31,006 and 4,016,043, while optional procedure D is known as the "double antibody", or "DASP" procedure.

A further alternate diagnostic procedure employs multiple labeled compounds in a single solution for simultaneous radioimmune assay. In this procedure disclosed in U.S. Patent No. 4,762,028 to Olson, a composition may be prepared with two or more analytes in a coordinated compound having the formula: radioisotope-chelator-analyte.

In each instance, the advanced glycoslation endproduct forms complexes with one or more binding partners and one member of the complex may be labeled with a detectable label. The fact that a complex has formed and, if desired, the amount thereof, can be determined by the known applicable detection methods.

With reference to the use of an AGE antibody as a binding partner, it will be seen from the above that a characteristic property of Ab ₂ is that it will react with Ab,. This is because Ab, raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab ₂ . For example, Ab ₂ may be raised in goats using rabbit antibodies as antigens. Ab ₂ therefore would be anti-rabbit antibody raised in goats. Where used and for purposes of this description, Ab, will be referred to as a primary or anti-advanced

glycosylation endproduct antibody, and Ab ₂ will be referred to as a secondary or anti-Ab, antibody.

The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.

Suitable radioactive elements may be selected from the group consisting of ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ C1, ^5, Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, "ϊ, ^12S I, ^I3I I, and ¹⁸⁶ Re. In the instance where a radioactive label, such is prepared with one of the above isotopes is used, known currently available counting procedures may be utilized.

In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectro-photometric, thermometric, amperometric or gasometric techniques known in the art. The enzyme may be conjugated to the advanced glycosylation endproducts, their binding partners or carrier molecules by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Also, and in a particular embodiment of the present invention, the enzymes themselves may be modified into advanced glycosylation endproducts by reaction with sugars as set forth herein.

Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase, hexokinase plus GPDase, RNAse, glucose oxidase plus alkaline phosphatase, NAD oxidoreductase plus luciferase, phosphofructokinase plus phosphoenol pyruvate carboxylase, aspartate aminotransferase plus phosphoenol pyruvate decarboxylase, and alkaline phosphatase. U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternative labeling material and methods.

A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine and auramine. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.

The present invention includes assay systems that may be prepared in the form of test kits for the quantitative analysis of the extent of the presence of advanced glycosylation endproducts. The system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to a binding partner to the advanced glycosylation endproduct such as a receptor or ligand as listed herein; and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s) .

In a further embodiment of this invention, commercial test kits suitable for use by a medical specialist may be prepared to determine the presence or absence of advanced glycosylation endproducts. In accordance with the testing techniques discussed above, one class of such kits will contain at least labeled AGE, or its binding partner as stated above, and directions, of course, depending upon the method selected, e.g., "competitive", "sandwich", "DASP" and the like. The kits may also contain peripheral reagents such as buffers, stabilizers, etc.

For example, a first assay format contemplates a bound receptor to which are added the ligand and the analyte. The resulting substrate is then washed after which detection proceeds by the measurement of the amount of ligand bound to the receptor. A second format employs a bound ligand to which the receptor and the analyte are added. Both of the first two

formats are based on a competitive reaction with the analyte, while a third format comprises a direct binding reaction between the analyte and a bound receptor. In this format a bound receptor-specific carrier or substrate is used. The analyte is first added after which the receptor is added, the substrate washed, and the amount of receptor bound to the substrate is measured.

More particularly, the present invention includes the following protocol within its scope:

A method for determining the amount of advanced glycosylation endproducts in an analyte comprising:

A." providing a sample of monocytes bearing the present receptor protein(s); B. inoculating said sample with a known advanced glycosylation endproduct bound to a whole cell; and

C. counting the whole cells of Step B that are bound to and/or internalized by said sample.

The specific protocol set forth above is illustrated in the examples that follow later on herein, and reflects the broad ^* latitude of the present invention. All of the protocols disclosed herein may be applied to the qualitative and quantitative determination of advanced glycosylation endproducts and to the concomitant diagnosis and surveillance of pathologies in which the accretion of advanced glycosylation endproducts is implicated. Such conditions as diabetes and the conditions associated with aging, such as atherosclerosis and skin wrinkling represent non-limiting examples, and accordingly methods for diagnosing and monitoring these conditions are included within the scope of the present invention.

Accordingly, a test kit may be prepared for the demonstration of the presence and activity of AGEs, comprising:

(a) a predetermined amount of at least one labeled immunochemically reactive component obtained by the direct or

indirect attachment of an advanced glycoslation endproduct binding partner comprising the present receptor protein(s) or a specific binding partner thereto, to a detectable label; (b) other reagents; and (c) directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

(a) a known amount of a binding partner as described above, or a ligand thereof, generally bound to a solid phase to form an immunosorbent, or in the alternative, bound to a suitable tag, or plural such end products, etc. (or their binding partners) one of each;

(b) if necessary, other reagents; and

(c) directions for use of said test kit.

In a further variation, the test kit may comprise:

(a) a labeled component which has been obtained by coupling the above binding partner to a detectable label;

(b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand is selected from the group consisting of:

(i) a ligand capable of binding with the labeled component (a) ;

(ii) a ligand capable of binding with a binding partner of the labeled component (a) ;

(iii) a ligand capable of binding with at least one of the component(s) to be determined; and

(iv) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined; and

(c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction between the advanced glycosylation endproduct and the binding partner.

The present invention will be better understood from a consideration of the following illustrative examples and data.

Accordingly, Examples I and II presented in parent application Serial No. 453,958 confirm the basic hypothesis that the in vivo recognition and removal of AGEs is receptor mediated, and Examples III and IV present respectively, the investigations and experiments that have resulted in the identification of the liver membrane- and mesangial cell derived receptors of the present invention.

EXAMPLE I

In this example, the existence of the receptor-mediated clearance system of advanced glycosylation endproducts that underlies the present assay was initially explored, in part, by the performance of a competitive phagocytosis assay was conducted with whole monocytes. A full review of the details of the experimental procedures involved is presented in U.S. Patent No. 4,900,747, and reference may be made thereto for such purpose.

In this example, human red blood cells (RBCs) were collected and isolated, and separate quantities were prepared to facilitate the performance of the assay. Specifically, a quantity of RBCs were opsonized by incubation with an appropriate antiserum. A further quantity was bound to the advanced glycosylation endproduct FFI by a carbodiimide bond, and additional RBCs were separately glycosylated by reactions with glucose, glucose-6-phosphate, xylose and arabinose, respectively. Lastly, AGE-BSA and human monocytes were prepared. Phagocytosis assays proceeded by the incubation of the RBCs with the monocyte cultures followed by fixation of the sample wells and lastly counting under 4Ox phase microscopy.

An FFI-RBC half life assay was also conducted with Balb/c mice that were inoculated with FFI-RBC suspensions labeled with

51 Cr. The labeled cells were washed at least four times to remove unbound isotope. Twelve Balb/c mice were then injected

intravenously with 200 μl RBC suspension. Each sample was administered in three Balb/c mice. At appropriate time intervals the mice were bled (0.2 ml) and radioactivity levels were measured by counting.

RESULTS Maximum binding of red cells was observed on Day-7 of monocyte incubation in vitro. Maximum binding and endocytosis of FFI- RBC was complete within 30-45 minutes while opsonized cells were maximally bound within 15 minutes. At the end of one hour incubation of FFI-coupled RBCs with cultured human monocytes, per cent phagocytosis and phagocytic index were estimated. As shown in Figure 1, % erythrophagocytosis of FFI-modified red cells (55%) and IgG-coated red cells (70%) were significantly higher than that of control PBS-treated cells (4%) . Similarly the phagocytic index of FFI-treated RBCs was greatly elevated (3.4) as compared to normal controls (1.2) .

In order to establish the specificity of the interaction of FFI-RBCs with the human monocytes, competition experiments were carried out in which binding and ingestion of red cells was observed in the absence and presence of AGE-BSA, prepared as described in Methods (Vlassara et al., supra. ) . As shown in Figure 2, the addition of AGE-BSA at concentrations of 500 μg/ml inhibited the FFI-RBC binding by more than 70% of the control. In contrast, AGE-BSA did not inhibit opsonized or PBS-treated red cells, even at maximal concentrations (1 mg/ml) . These data suggested that FFI-modified red cells were recognized and bound specifically by the monocyte AGE-binding site, and consequently confirmed the operability of the present assay.

DISCUSSION The above tests extend previous observations on the recognition of advanced glycosylation endproducts (AGE) by a specific monocyte/macrophage receptor, and present evidence

that such adducts once attached chemically or formed in vitro on the surface of intact human cells can induce cell binding and ingestion by normal human monocytes. The experiments establish the development of a competitive receptor-based assay for AGEs measuring by way of illustration herein, AGE- red cell binding in the presence of large excess of AGE-BSA (Vlassara et al., supra.).

EXAMPLE II

This example comprises a series of experiments that were initially performed to measure the ability of agents to stimulate phagocytic cells to stimulate uptake and degradation of endproducts (AGEs) , and thereby further confirmed the hypothesis that this activity is receptor-mediated.

Several AGEs were prepared using the same procedure as disclosed in Example I, above. Accordingly, FFI-HA was prepared as described and quantities were bound to both human and bovine albumin. A water soluble carbodiimide was used to attach the acid moiety of the FFI-HA to an amino group on the protein. After preparation, the conjugate was purified and then used in vitro to stimulate macrophages, by incubation for from 4 to 24 hours.

The AGEs that were to be observed for uptake and degradation were appropriately radiolabeled so that they could be traced. Thereafter, the stimulated macrophages were tested by exposure to the radiolabeled AGEs following exposure to various agents to measure the effect that these agents had on the ability of the macrophages to take up and degrade the labeled AGEs. The above procedures conform to the protocol employed by Vlassara et al., supra. and confirmed that a competitive assay based on a cellular receptor for AGEs is feasible.

It was also demonstrated that monocyte or macrophage cells can also be stimulated by AGE-carrier molecules which result in

cells with enhanced ability to bind, internalize and degrade other AGE-molecules. AGE-carrier molecules are made, for example, from the reaction of glucose or glucose-6-phosphate with albumin. After purification of the reaction product, the AGE-albumin uptake of AGE-macromolecules demonstrated as in (A) above. AGE-BSA (prepared from the incubation of glucose- 6-phosphate with albumin for 6-8 weeks) at 0.1 mg/ml demostrates a stimulatory effect on AGE-BSA uptake by human monocytes (Figure 4, bar AA) , and shows a slight stimulation at higher concentrations (bars BB and CC) . This observation further supports the role of these ligands in conjunction with cellular receptors and point to the application of these agents in a competitive AGE assay protocol.

EXAMPLE III

The following example discloses the purification from rat liver, and partial amino acid sequencing, of two membrane proteins of approximately 60 and 90 kD, respectively, that bind AGE-modified proteins. Both of the proteins presented are expressed on the surface of rat monocytes and resident peritoneal macrophages, suggesting a relationship to the AGE- receptor system earlier identified on these cells. The proteins are believed to be involved in tissue repair and remodelling.

MATERIALS AND METHODS

Chemicals and reagents. Bovine serum albumin (BSA) (Fraction

V) , bovine ribonuclease, glucosamide-BSA, glucose-6-phosphate and collagen I were purchased from Sigma Chemical Co. (St.

Louis, MO) . Triton X-114 and Triton X-100 were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN) . Sodium ¹²⁵ iodide was obtained from New England Nuclear (Boston, MA) . Nitrocellulose membranes were from Schliecher & Schuell (Keene, NH) . CNBr-activated Sepharose 4B was purchased from Pierce (Rockford, IL) . LDL and acetyl-LDL were the generous gift of Dr. David Via (Baylor College of Medicine, Houston,

TX) . 2-(2-furoyl)-4(5)-(2-furanyl)-lH-imidazole (FFI) and its derivative, FFI-1-hexanoic acid (FFI-HA) were kindly provided by Dr. Peter Ulrich (Geritech Inc. , Northvale, NJ) .

Preparation of AGE-proteins and formaldehyde-modified proteins. AGE-BSA, AGE-RSA, AGE-ribonuclease and AGE-collagen I were generated by standardized methods as previously described in detail. In brief, each protein solution (25 mg/ml) was incubated with either 0.5 M glucose or glucose-6-phosphate in 100 mM phosphate buffer (pH 7.4) at 37°C for six weeks under sterile conditions, and then low molecular weight reactants were removed by dialysis against phosphate buffered saline (PBS; 20 mM sodium phosphate buffer containing 0.15 M NaCl, pH 7.4). FFI-HA was coupled to BSA with the water-soluble carbodiimide, EDAC, as described previously. Characteristic fluorescence of the AGE-proteins was observed at 450 nm upon excitation at 390 nm. Formaldehyde-modified BSA was prepared as described, by incubating BSA in 0.1 M sodium carbonate buffer (pH 10) with 0.33 M formaldehyde at 37°C for 5 hours, followed by extensive dialysis against PBS. All protein concentrations were determined by the method of Bradford. Protein and AGE-protein preparations were radio¬ labeled with [ ^I25 I] by the Iodo-Gen method for example, 2 mg of AGE-BSA were incubated with 25 mCi carrier-free [ ¹²⁵ I] in an Iodogen-coated glass vial at room temperature for 45 min. In order to separate free from bound [ ^l25 I] , the sample was fractionated by Sephadex G-25M column chromatography and dialysis against PBS, until at least 98% of the ¹²⁵ I was trichloroacetic acid-precipitable. The specific radioactivity of the labeled ^I25 I-AGE-BSA was between 8,000-15,000 cpm/ng protein.

BSA and AGE-BSA Sepharose were prepared by reacting either BSA or AGE-BSA (25 mg/ml) with CNBr-Sepharose gel (5 ml/g of dry powder) according to the manufacturers instructions, in coupling buffer (0.1 M NaHC0 ₃ , 0.5 M NaCl, pH 8.3). The mixture was rotated for 2 h at room temperature. Excess

ligand was washed from the resin with coupling buffer and then with Tris-HCl buffer (0.1 M, pH 8.0) for 2 h at room temperature to block remaining active groups. The resin was then washed with three cycles of sodium acetate buffer (0.1 M, pH 4) containing NaCl (0.5 M) , followed by Tris-HCl buffer (0.1 M, pH 8) containing NaCl (0.5 M) , and stored at 4-8°C.

In Vivo Studies. For tissue distribution studies, AGE-rat serum albumin (AGE-RSA) was prepared as described above, and radioiodinated to a specific activity of 8.3 X 10 ⁵ cpm/μg.

Similarly, normal RSA was iodinated to a specific activity of 6.2 X 10 ⁵ cpm/μg. Freshly drawn rat red blood cells (RBC) were labeled with ⁵¹ Cr to allow subsequent correction for tissue counts for blood-associated radioactivity. Approximately 1 mCi of ⁵¹ Cr was added to 10 ml RBC, mixed thoroughly and allowed to incubate for 30 min at room temperature prior to washing with PBS containing 1% BSA and 0.1% dextrose until supernatant radioactivity was less than 1% of that in the packed RBC. Buffer was added to labeled RBC to obtain a hematocrit of 40% and the labeled RBC were used immediately.

To determine the tissue distribution of AGE-ligands, normal male Sprague-Dawley rats (200 g) were divided into groups of five, and anesthetized with sodium pentobarbital (40 mg/kg) . All animals received an injection of ^5l Cr-RBC (0.65 ml, i.v.) five minutes prior to the labeled ligand. The indicated groups of rats received either ^12S I-AGE-RSA (50 μg in 0.1 ml, i.v.) or an identical amount of ¹⁵ I-normal RSA. At the indicated time intervals, 0.5 ml aliquots of blood were drawn and various organs were removed and counted for radioactivity. The specificity of AGE-ligand uptake in various organs was assessed by injecting groups of animals with excess non- labeled AGE-BSA (5 g) two minutes prior to administration of 50 μg of ¹²⁵ I-AGE-BSA. After 5, 20, and 60 minutes, 0.5 ml blood samples were collected, animals were sacrificed, and various organs and tissues were collected and counted for radioactivity. The red blood cells were lysed with water, and

protein was precipitated with 20% TCA. The organs were weighed, homogenized with a hand homogenizer, protein was precipitated with 20% TCA and counted for radioactivity. The tissue-to-blood isotope ratio (TBIR) was calculated by the formula TBIR=[ ¹²⁵ I/ ⁵¹ Cr in tissue]/[ ¹²⁵ I/ ⁵¹ Cr in blood]. TBIR is a dimensionless index of the degree to which any tissue sample has sequestered labeled ligand relative to the blood.

Solubilization and fractionation of hepatic membrane proteins. Liver membranes were prepared according to the method of Thorn et al. with some modifications. For a typical membrane preparation, 14 grams of rat liver were homogenized in 80 ml TNE buffer [50 mM Tris-HCl buffer (pH 8.0), containing 150 mM NaCl, 0.1 mM EDTA, and 23 μg/ml phenylmethylsulfonyl fluoride (PMSF) ] and centrifuged for 10 min at 3000 x g. The supernate was layered on top of a solution of 40% sucrose in TNE buffer, and centrifuged at 24,000 x g for 1 h at 4°C. The membranes were collected from the interface with a Pasteur pipette. The membrane preparation was solubilized in TNE buffer containing 2% Triton X-114 at 4°C and clarified by centrifugation for 30 min at 100,000 x g. The supernate was then warmed to 30°C and the detergent phase, aqueous phase and detergent-insoluble pellet were separated according to the phase separation method described by Bordier. The resulting detergent phase was either used directly for purification of AGE-binding proteins or diluted 1:10 with PBS containing 2% Triton X-100 and 2 mM PMSF. This material (D-phase) was frozen at -80°C until further use.

In Vitro solid-phase AGE binding assay. AGE-binding activity was determined by a modified version of solid-phase binding assay developed for the IL-1 receptor. Aliquots of detergent-solubilized membrane proteins were blotted onto grid-marked nitrocellulose (NC) membranes. The blots were dried at room temperature and could be stored at room temperature for several weeks without apparent loss of binding

activity. The NC membranes were cut into small squares (0.9 cm ² ) with the immobilized protein at the center and distributed in 24-well trays (Costar, Cambridge, MA) . Immobilized protein was reconstituted in PBS, pH 7.4, containing 0.5% Triton X-100 for 30-60 min at room temperature. The blots were subsequently incubated in blocking buffer (PBS, pH 7.4, containing 2% BSA, 0.2% Triton X-100 and 1 mM MgCl ₂ ) for 2 h with agitation at 4°C. Specific ligand binding was carried out by adding 50-100 nM ¹²⁵ I-AGE-BSA directly to the blocking buffer and agitating at 4°C for a further 1.5 h. The NC membranes were transferred to a new tray and rinsed quickly 3 times with PBS containing 0.2% Triton X-100, followed by two additional 10 min. washings with PBS. Ligand binding was then evaluated by autoradiography or gamma counting.

Ligand blotting. SDS-PAGE and electro-blotting were performed as previously described. Proteins were electrophoretically separated on either 8-16% or 4-20% gradient polyacrylamide gels. After electro-blotting the proteins from the gel onto NC membranes, the blots were washed at 4°C overnight with PBS containing 0.2% Triton X-100. The blots were then incubated in blocking buffer for 3 h at 4°C with agitation. Ligand binding was performed by adding 10 nM ^l25 I-AGE-BSA to the blocking buffer. After 1.5-2 h at 4°C the blots were washed three times with blocking buffer for 1 min each and two times for 10 min each. After air drying, the ligand binding was evaluated by autoradiography.

Isolation of p60 and p90 from rat liver membranes. Unless otherwise indicated, all purification procedures were performed at 4°C. The detergent phase of the rat membrane preparation (D-phase, -860 g protein) was applied to a polyethylenimine cellulose column (PEI) (3 X 30 cm) equilibrated with TNE buffer. After washing with equilibration buffer (TNE plus 2 mM PMSF containing 1% CHAPS) , the proteins bound to the PEI column were eluted by a 240 ml linear gradient of 0.1 - 1.5 M NaCl in the equilibration

buffer. Fractions were analyzed for binding activity by the solid-phase AGE binding assay. The active fractions were pooled and dialyzed overnight against TNE buffer containing 1% CHAPS. The PEI pool (160 mg protein, 80 ml) was then applied to a DEAE-cellulose column (2.5 x 20 cm) previously equilibrated with TNE/CHAPS/PMSF equilibration buffer. The column was washed with 4 column volumes of equilibration buffer and proteins were eluted by a 200 ml linear gradient of 0.2-1.5 M NaCl in equilibration buffer. The fractions which contained AGE-BSA binding activity were pooled and concentrated by ultrafiltration (Centricon 10, Amicon) . The concentrated DEAE pool (80 mg protein) was cycled three times through a BSA-Sepharose 4B column (2 x 12 cm, 10 mg of BSA per ml of gel) , to eliminate proteins which bound to BSA. The flow-through from this BSA-Sepharose column was then applied to an AGE-BSA-Sepharose 4B column (2 x 6 cm, 10 mg of AGE-BSA per ml of gel) and cycled twice. The column was washed with .25 column volumes of PBS buffer, pH 7.4, containing 0.2% Triton X-100, and 1 mM PMSF. The proteins bound to the AGE- BSA column were eluted with the step-wise addition of PBS buffer containing 1.5 M NaCl, 0.2% Triton X-100, and 1 mM PMSF. Each fraction was dialyzed against PBS containing 1 mM PMSF, concentrated by ultrafiltration (Centricon 30, Amicon) and analyzed for ¹²⁵ I-AGE-BSA binding activity by the solid- phase binding assay.

Preparative electrophoresis was performed as described in detail elsewhere. In brief, 50 μg of the protein mixture which had been affinity purified over AGE-BSA was boiled for three min in sample buffer (0.03 M Tris-HCl, pH 6.8, 1% SDS, 5% glycerol, 0.015% Bromophenol Blue) in the presence of 0.1 M 2-mercaptoethanol and electrophoresed through 10% polyacrylamide gels in the presence of 0.1% SDS. The 60 kD and 90 kD protein bands were excised and electro-eluted (Elutrap, Schleicher & Schuell) in Tris buffer (25 mM, pH 8.5) containing glycine (192 mM) and 0.1% SDS, as described.

Antibody generation. Laying hens were injected subcutaneously at multiple sites with a total of 100-150 μg of electrophoretically purified p60 or p90 in complete Freund's adjuvant (Pocono Rabbit Farm and Laboratory, Canadensis, PA.). On day 14 and 21 the hens were injected with an additional 60 μg of each protein in complete Freund's adjuvant. Further boosts of 80-100 μg of the corresponding proteins in incomplete Freund's adjuvant were given one month after the initial series. Eggs and serum from the chickens immunized with p60 or p90 proteins were separately collected.

Immunoglobulins from the yolks were extracted according to the method of Poison et al., while serum immunoglobulins were isolated by a combination of ammonium sulphate (30%) precipitation and DEAE cellulose chromatography.

Western Blotting. Ten μg aliquots of detergent-solubilized samples of membrane protein were boiled in sample buffer in the presence of 0.1 M 2-mercaptoethanol, and electrophoresed through gradient gels (8-16%) . After transferring onto nitrocellulose, the membranes were rinsed in TBS-t buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween 20) and blocked with TBS-t buffer containing 2% BSA for 1 hour at 4°C. The blots were probed with either anti-60 kD or anti-90 kD avian IgG or pre-immune chicken IgG (10 μg/ml) for 60 min at 4°C and then washed with TBS-t buffer three times, for 5 min each. The blots were then incubated with goat anti-chicken alkaline phosphatase conjugate (1:1000 dilution) for 1 hr at 4°C. Color development was achieved according to instructions of the manufacturer (Promega, Cleveland, OH) .

Flow Cvtometric analysis

Cell preparation. Heparinized blood was drawn from male Sprague-Dawley rats (200-300 g) by cardiac puncture. Purified monocytes were prepared over Ficoll-Hypaque and Percoll gradients. Resident rat peritoneal macrophages were obtained from rats by washing the peritoneal cavity with 20 ml of PBS and were characterized by flow cytometry.

Fluorescence Flow Cytometry. The expression of p60 and p90 AGE-binding proteins on rat monocytes and macrophages was determined by indirect immunofluorescence. Single color cell staining was performed by incubating one million cells with biotinylated anti-p60 or anti-p90 primary antibodies at a final concentration of 5 μg/ml for 20 min at 4°C. Cells were washed in staining buffer (PBS, 3% FBS, 0.1% NaN ₃ ) and then incubated with FITC-conjugated avidin (Becton Dickinson, Mountain View, CA) . Background fluorescence was determined by staining the cells with a relevant isotypic control antibody, biotinylated chicken IgG used in identical concentrations (5 μg/ml) . Cells were analyzed using a FACSCAN (Becton Dickinson, Mountain View, CA) with gates set by forward angle light scatter and side scatter. Fluorescence emission for FITC was detected by selectively collecting at 500-537 nm on at least 5000 labeled cells, gated to include monocytes/macrophages and to exclude lymphocytes, other non- monocytic cells and dead cells. The data were analyzed by Paint-A-Gate software (Consort 30, Becton Dickinson, Mountain View, CA) .

For the cross-competition study, 10 ⁶ rat monocytes were treated with either 5 μg/ml biotinylated anti-p60 antibodies in the presence of 20-fold excess anti-p90 antibodies, or 5 μg/20 μl biotinylated anti-p90 in the presence of 20-fold excess anti- p60. The antibody-treated cells were then labeled using FITC- avidin and analyzed by flow cytometry.

RESULTS

In vivo tissue distribution of AGE-binding activity. Applicants previously identified a 90 kD protein on mouse and human monocytes/macrophages which selectively binds AGE- proteins. Since these sources are not convenient to provide sufficient material for further biochemical characterization, alternate tissue sources were sought. As a first step, the distribution of AGE-specific binding activity in rat tissues

was examined by uptake studies of ¹²⁵ I-AGE-RSA. Either ^,5 I-AGE- RSA (50 μg, 8.3 X 10 ⁵ cpm/μg) or ^I25 I-normal RSA (50 μg, 6.2 X 10 ⁵ cpm/μg) was injected intravenously into rats along with ⁵¹ Cr-labeled RBC, as described in Methods. After 10 minutes, greater than 50% of the AGE-RSA was concentrated in the liver, whereas the liver uptake of non-modified ^l25 I-RSA was consistently less than 10% of the AGE-RSA values (Figure 5A) . Tissue accumulation of AGE-RSA was not affected by the prior injection of 100-fold excess non-labeled RSA (5 mg, not shown) . In contrast, pre-treatment of rats with excess non- labeled AGE-RSA (5 mg) decreased the accumulation of AGE-BSA in the liver by about 45% after 10, 20 and 60 minute intervals (Figure 5B) . The uptake of AGE-RSA remained uniformly low in all other major organs, with or without the non-labeled competitor. It was apparent that the liver had a high specific capacity to accumulate AGE-protein and therefore represented a potentially rich source for the isolation of the AGE-binding proteins.

l. AGE-binding assay

To facilitate the isolation of the AGE-binding proteins from liver a solid-phase assay system was developed involving the immobilization of detergent-solubilized membrane proteins onto nitrocellulose and probing for ligand-specific binding activity with ^I25 I-AGE-BSA as described in Methods. Figure 6A shows the effect of increasing amounts of crude liver membrane proteins on AGE-ligand binding. Total AGE-BSA binding increases in proportion to the amount of membrane proteins immobilized on the filters, whereas non-specific binding was negligible. The AGE-binding kinetics of these membrane proteins after blotting onto nitrocellulose is shown in Figure 6B. When -8 μg of hepatic membrane proteins immobilized onto NC filters were incubated with increasing amounts of ¹²⁵ I-AGE- BSA, saturable binding was observed with a B _nιax of 0.22 pmol/8 μg of protein (Figure 6C) . A dissociation constant (K _d of 4 X 10 ^"8 M) was revealed by Scatchard analysis of the binding data.

The specificity of liver cell binding activity for AGE adducts on protein was determined in competition experiments testing ¹²⁵ I-AGE-BSA against several different AGE-protein competitors, as well as against ligands known to bind to other scavenger receptors (Figure 6D) . The addition of 150-fold excess AGE- RNAse or AGE-collagen I completely inhibited the binding of radio-labeled AGE-BSA to crude hepatic membrane protein extracts immobilized on NC filters. In contrast FFI-BSA, formaldehyde-treated BSA, glucosamide-BSA (a chemically linked glucose-BSA compound) or acetyl-LDL did not compete against the binding of labeled AGE-BSA.

Using similar detergent-solubilized membrane preparations from heart, kidney, brain, or lung obtained by identical procedures as described for liver, ^I25 I-AGE-BSA specific binding activity was examined by the same solid-phase AGE-binding assay. Liver membrane proteins exhibited the highest binding activity among all tissues examined, consistent with our in vivo observations (data not shown) .

2. Purification of rat liver AGE-binding proteins Using the solid-phase AGE binding assay as a means of monitoring AGE-binding activity column fractions, the isolation of AGE-binding protein(s) was pursued by the procedure outlined in Table 1 and described in detail in

Materials and Methods. In brief, rat liver membrane proteins were solubilized in Triton X-114. After detergent phase- separation, the D-phase was subjected to chromatography on PEI-cellulose, DEAE-cellulose, BSA-Sepharose, and finally, AGE-BSA-Sepharose. After elution from the AGE-BSA Sepharose column, the fractions were assessed for AGE-binding activity by the solid-phase AGE binding method (Figure 7) .

Analytical SDS-PAGE electrophoresis of the active fractions obtained from the AGE-BSA-column revealed the presence of two main protein bands with approximate molecular weights of 60 kD (p60) and 90 kD (p90) (Figure 7, inset). In order to separate

larger amounts of these AGE-binding proteins, AGE-BSA column eluate fractions were subjected to preparative PAGE and the individual proteins were separately electro-eluted from respective gel slices.

Gel-purified p60 and p90 were blotted onto Immobilon membranes and amino terminal sequences were obtained at the Rockefeller University sequencing facility. Table 7 records the N- terminal sequence obtained from each of these proteins, which data is also presented herein in Figures 11 and 12.

Comparison of these sequences with the translated Genbank database did not reveal significant similarity to other known proteins.

Ligand blotting of p60 and p90 proteins immobilized on nitrocellulose, using ^I25 I-AGE-BSA as probe, revealed that only the 60 kD protein bound this ligand (not shown) . After blotting on nitrocellulose, the 90 kD protein did not bind ^12S I- AGE-BSA, although p90 did bind to the AGE-BSA-Sepharose matrix and was not retained on the BSA-Sepharose column.

When crude rat liver membrane proteins (D-phase) were separated by SDS-PAGE under non-reducing conditions, transferred to NC filters, and probed for ligand binding with ^I25 I-AGE-BSA, a single major AGE-BSA binding band at an approximate molecular mass of 60 kD was revealed (Figure 8A, lane 1) . The binding of this protein to AGE-BSA was inhibited partially in the presence of a 25-fold excess (lane 2) and completely by a 150-fold excess of non-radioactive AGE-BSA (lane 3) . No other prominent bands were observed under these conditions. It thus appears likely that nitrocellulose immobilization may inactivate the binding properties of p90, or that p90 is a p60-associated protein which lacks independent AGE-binding activity.

3. Immuno-characterization of AGE-binding proteins Purified p60 and p90 proteins were injected into chickens in

order to obtain specific polyclonal antibodies. Preparation of avian IgG specific for each of the proteins were isolated either from egg yolk or serum as described in Materials and Methods. The specificity of each of the antibodies was verified by Western blot analysis, using the same crude liver membrane protein extract analyzed above by ligand blotting (Figure 8B) . The antibody to the p60 AGE-binding protein recognized a major protein band at approximately 60 kD (lane 2) , while the pre-immune IgG did not (lane 1) . Similarly the antibody to p90 recognized a single protein band at about 90 kD (lane 4) , whereas the pre-immune antibodies did not (lane 3).

The antibodies to p60 and p90 were used to screen for the expression of these proteins on the surface of rat peripheral blood monocytes and peritoneal macrophages. FACS analyses which demonstrated the presence of each protein on the surface of both cell types are shown in Figure 9. Panels A and B illustrate flow cytometric detection of p60 and p90 on the surface of rat monocytes. Figure 9 also shows that the binding of either anti-p60 or anti-p90 to the monocyte cell surface was not affected by a 20-fold excess of the heterologously directed antibody (panels C and D respectively). Distinct binding of the anti-p60 and anti-p90 antibodies was also observed when the rat peritoneal resident macrophages were analyzed by flow cytometry (Figure 9, panels E and F, respectively) . A small subgroup of highly fluorescent cells of an unspecified nature with a non-specific FITC staining pattern was also noted, using antibody as well as isotypic controls.

In order to confirm that p60 and p90 were both AGE-binding proteins expressed independently on rat peritoneal resident macrophages, ¹² I-AGE-BSA binding inhibition experiments were carried out using each antibody separately (undiluted: 10 μg/ml) as well as in combination (Figure 10A) . In the presence of increasing concentrations of anti-p60 antibody

significant AGE-BSA binding inhibition was observed (up to 80% at a final concentration of 10 μg/ml) . Similarly ¹²⁵ I-AGE-BSA binding was inhibited up to 60% by the anti-p90 antibody, while the combination of both antibodies at a dilution of 1:10 provided 84% inhibition. When radio-labeled FFI-BSA (made from the chemically synthesized model AGE compound, FFI, crosslinked onto BSA with a carbodiimide reagent) was used as the ligand, anti-p60 as well as anti-p90 mediated a concentration-dependent inhibition of FFI-modified BSA binding (Figure 10B) . Moreover, and as found with AGE-BSA, a combination of anti-p60 and anti-p90 antibodies exerted greater inhibition compared to either anti-p60 alone or anti- p90 alone. No inhibitory effect was noted when isotypic control antibodies were used, even at the maximal concentration, in conjunction with either modified BSA ligand (not shown) .

TABLE I: Purification of Rat Liver AGE Binding Proteins

^,25 I-AGE-BSA Binding

Rat liver membrane proteins were procured and prepared as described. Specific binding of AGE- BSA was determined by the solid phase binding assay.

Binding activity could not be determined due to the presence of SDS.

TABLE II: N-Terminal Amino Acid Sequence Analysis of AGE-Binding Proteins from Rat Liver

60 kD: XGPRTLVLLDNLNVRDTHXLFF

90 kD: XEVKLPDMVSLXD

X INDICATES UNIDENTIFIED RESIDUES.

DISCUSSION The above experiments confirm the isolation and discovery of two novel rat liver membrane proteins, designated p60 and p90 by their migration SDS-PAGE, which specifically bind to protein ligands modified by advanced glycosylation endproducts (AGEs) . Amino terminal sequence analysis indicates that these proteins bear no significant homology to each other nor to any previously sequenced proteins currently available in the Genbank database. Both p60 and p90 are present on rat monocytes and macrophages and are immunoreactively and functionally distinct. Of importance is the fact that these binding proteins have been distinguished from both the recently reported macrophage scavenger receptor for acetyl-LDL, a functional trimer composed of three 77 kD glycoprotein subunits; and from the binding proteins for formaldehyde- treated albumin with M _r 's of 30 and 52 kD.

The p60 and p90 AGE-binding proteins were isolated from rat liver, upon the determination that this organ acts as a major filter for the in _. vivo clearance of AGE-modified macromolecules; liver presents the highest capacity to specifically sequester AGE-proteins administered intravenously (Figure 5) . An isolation procedure, including elution of detergent-solubilized membrane proteins from an AGE-protein affinity matrix was devised, and the p60 and p90 AGE-binding proteins were found to

co-purify. When immobilized on nitrocellulose, however, only p60 retained binding activity for AGE-modified ligands. Just as they co-purify over anion exchange and ligand affinity columns, p60 and p90 were also observed to co-purify in hydroxylapatite chromatography (not shown) . The extent of this co-purification continues to be examined.

The liver is a complex organ containing several cell types, including macrophages and endothelial cells, both of which have been shown to bear AGE-receptors. To determine whether macrophages also expressed a 60 kD AGE- binding protein, and whether there was any relationship between the macrophage 90 kD and the liver p90 AGE- binding proteins, specific polyclonal antibodies to liver p60 and p90 were developed.

The specificity of these antibodies was demonstrated by Western analysis of crude liver membrane extracts, revealing that each antiserum identified a single protein band of the appropriate molecular weight. Flow cytometric analysis of rat peripheral monocytes and peritoneal resident macrophages revealed that each antisera bound to the surface of both of these cell types. Cross-competition studies performed on monocytes revealed no cross-reactivity between the two antibodies. These data indicate that the p60 and p90 AGE-binding molecules originally isolated from whole liver preparations are each present on monocytes as well as macrophages (Figure 9) .

Flow cytometric binding inhibition experiments clearly demonstrated that p60 and p90, expressed on the surface of monocytes/macrophages, independently bound AGE- modified ligands. Interestingly, a combination of antibodies specific for p60 and p90 mediated greater

inhibition of AGE-protein binding than did either antibody alone.

Either antiserum, used independently or in combination, prevented more than 90% of binding FFI-BSA to rat macrophages. In the case of p60, this finding is surprising given that this binding protein does not bind FFI-BSA in a solid-phase ligand blotting assay. With regard to liver p90, the flow cytometry and FFI-binding inhibitory data indicate that this molecule may be closely related to the 90 kD protein isolated from murine RAW 264.7 cells. In fact, preliminary experiments using antibodies raised against rat liver p60 and p90 proteins to stain mouse RAW cells provided flow cytometric and binding inhibition results similar to those obtained with rat monocytes and macrophages, strongly supporting a structural similarity between the AGE-binding proteins of these two rodent species.

EXAMPLE 3

This example presents the investigations and experiments that have resulted in the identification of the mesangial cell-derived AGE receptors of the present invention.

MATERIALS AND METHODS

Cell culture: Primary cultures of rat MCs were obtained from outgrowths of isolated rat glomeruli by Dr. M. Ganz (Yale University, N.Haven, Ct.) . In brief, rats were anesthetized with ether and the kidneys were excised under sterile conditions. After removing the kidney capsule, the kidney cortices were isolated, minced to a fine paste with a razor blade, and then pressed through serial stainless steel sieves (Tyler USA No. 140, 80, and 200) . Glomeruli were collected from the top of the 75 micrometer sieve. This process resulted in >98% pure glomeruli. The glomeruli were then pelleted and

resuspended in DMEM supplemented with 20% FBS, 5 μg/ml bovine insulin, 2 mM L-glutamine, and 40 μg/ml gentamicin. The glomerular suspensions were plated onto tissue culture flasks and incubated at 37°C in 5% C0 ₂ . Primary cultures were allowed to grow for 3-4 weeks at which time the MCs were confluent. MCs were used between the fourth and ninth passages.

The purity of the rat MC populations was documented. The MCs exhibited a uniform straplike appearance and stained positively for Thy 1-1 antigen, myosin and actin. They were sensitive to mito ycin C, a MC toxin, but were resistant to the aminonucleoside puromycin, an epithelial cell toxin. Fibroblast contamination was excluded by demonstrating the ability of the cells to grow in media in which L-valine had been substituted for D-valine. In addition, they stained negative for factor VIII and cytokeratin. Over the experimental period they continued to maintain a uniform stellate appearance.

Human MC were provided by Dr. J. Floege and Dr. K. Resch, (Hannover, FRG) . In brief, normal human kidney tissue was obtained from nephrectomy specimens. Renal cortices were homogenized and glomeruli were isolated following passage through a series of graded sieves. The glomeruli were then treated with bacterial collagenase (Worthington Biochemical Corporation, Freehold, NJ) at 37°C for 30 minutes, and after extensive washing the glomerular remnants were plated onto tissue culture flasks in RPMI 1640, supplemented with 20% FBS, 2 mM L-glutamine, 2 mM sodium pyruvate, 5 μg/ml bovine insulin, 5 μg/ml human transferrin, 1% (v/v) non-essential amino acids, and gentamicin. Cellular outgrowths appeared between days 5-8, and all experiments were performed using cells between the fourth and tenth passage.

The purity of the MC population was demonstrated. In brief, immunofluorescent staining demonstrated prominent intracellular staining for smooth muscle cell myosin, MHC class I antigen, vimentin, collagen IV, and fibronectin. The cells stained negative for Fc-receptor, MHC II surface antigen, cytokeratin and factor VIII, and were able to grow in D-valine substituted medium.

Preparation of ligands: AGE- bovine serum albumin (BSA) and AGE-ribonuclease were made by incubating BSA and bovine ribonuclease (Sigma Chemical Co, St. Louis, MO) with 0.5 M glucose-6-phosphate (G6P) , at 37°C for 4 to 6 weeks in a 10 mM PBS buffer, pH 7.4 , in the presence of protease inhibitors and antibiotics. Unincorporated glucose was removed by dialysis against IX PBS. The concentration of AGE-BSA was determined and the concentration of ribonuclease was determined spectrophotometrically.

AGE formed on either BSA or ribonuclease was assessed based on characteristic absorption and fluorescence spectra (emission at 450 nm, excitation at 390 nm) and quantitated by a radioreceptor assay using intact RAW 264.7 cells grown in 96-well plates. According to this assay, AGE-BSA contained approximately 70 AGE U/mg (one unit of AGE is defined as the concentration of unknown agent required to produce 50% inhibition of standard ^,25 I- AGE-BSA binding) and AGE-ribonuclease contained 62 AGE U/mg.

Borohvdride Reduction: To examine the effect of early glycosylation product reduction on ligand binding, AGE-BSA was incubated with 200 molar excess NaBH ₄ (Sigma Chemical Co) for 10 minutes at 4°C, followed by 1 hour at room temperature. The reduced AGE-BSA was then dialyzed against IX PBS and the protein concentration was determined as above. The chemically defined AGE,

2-furoyl-4-(5)-(2-furanyl)-l-H-imidazole (FFI-HA), was synthesized and linked to BSA with 100 mM water soluble carbodiimide.

Iodination of AGE-BSA: AGE-BSA was iodinated with carrier-free-' ²⁵ I by the IODO-GEN method (Bio-Rad) of Fraker and Speck. Samples were dialyzed against PBS until >95% of radioactivity was trichloroacetic acid (TCA)-precipitable and the samples were iodide free.

Preparation of AGE-matrices: 6-well plates coated with rat tail collagen, type I, human fibronectin, and polylysine were purchased from Collaborative Research, Inc. (Bedford, MA) . AGE-matrices were produced by incubating the various matrix coated plates in 0.5 M G6P, at 37°C for 2-3 weeks in 10 mM PBS buffer (pH 7.4), as described for AGE-BSA. Control matrices were incubated under identical conditions in buffer alone. Following incubation, the plates were washed extensively with IX PBS. The amount of adhered collagen I was determined using a hydroxyproline assay, while adhered fibronectin and laminin were determined by the method of Lowry et al. after dissolving the matrix in 2 N NaOH at 37°C overnight, as described by Jones et al., and by absorbance at 280 nm. In both cases similar amounts of unmodified or AGE-modified matrix proteins adhered (collagen I, -85%; fibronectin, -70 %; laminin, -80% of the plated amount remained attached to the plates) . AGE levels in matrix proteins were quantitated by an AGE- specific radioreceptor assay, as described above. AGE- collagen I contained 47 AGE U/mg, AGE-fibronectin 54 AGE U/mg and AGE-laminin, 51 U/mg. Unmodified matrices contained less than 5 AGE U/mg protein.

Membrane preparation: Rat and human MCs were grown to confluency in 150 mm petri dishes. Cells were detached from the plates by PBS containing 3% EDTA and protease

inhibitors (2 mM phenyl ethylsulfonyl-fluoride [PMSF] , 10 μg/ml aprotinin, 5 ng/ml pepstatin, and 1 mM 1,10- phenanthroline) .

Following centrifugation, cells were disrupted with a tight Dounce homogenizer, pestle A, in a solution of PBS, with 10 mM EDTA and protease inhibitors, as stated above. The nuclear and organelle-enriched fraction were removed by centrifugation at 13,000 X g. Membranes were then isolated from the supernatant by centrifugation at

100,000 X g for 1 hour at 4°C. The resulting enriched membrane fraction was solubilized in PBS, containing 1% triton X-100, and 2 mM PMSF. The protein concentration was determined. This material was then used for binding and ligand blotting studies.

Binding studies: Filter binding studies were performed according to the method of Schneider et al. and Daniel et al. with minor modifications. 10-20 μg of MC membrane protein was dot-blotted onto nitrocellulose filters. The nitrocellulose filters were then cut and each dot was placed in a separate well of a 24-well plate. Following blocking of the filters for 1 hour at 4°C in PBS containing 1.5% BSA, binding studies were initiated by adding various concentrations of radioactive ligand to the individual wells.

At 2 hours, the nitrocellulose filters were washed 3 times with ice-cold IX PBS, and radioactivity bound was quantitated using a Packard Tricarb Scintillation counter.

Specific binding was defined as the difference between total binding (radioligand incubated with membrane protein alone) , and nonspecific binding (cells incubated with radiolabelled ligand plus 100-fold excess unlabelled ligand) . Scatchard analysis of the data was performed to

determine the binding affinity constant and the receptor number. Competition studies were performed in a similar manner to the binding studies, with the exception that the nitrocellulose filters were preincubated with the competitor for 1 hour before adding the radiolabelled ligand.

Binding studies were also performed on confluent MCs in 6-well plates. The studies were performed in 1 ml of RPMI-1640 at 4°C following the addition of various concentrations of radioactive ligand. After 2 hours of binding, the radioligand-containing medium was removed, and the cells were washed with ice-cold PBS. The cell monolayer was then disrupted with 1% triton X-100, and the cell-associated radioactivity was quantitated.

Protein concentration was determined by the method of Bradford. Specific binding was determined in an identical manner to that described above for the filter binding assay.

Ligand blotting: MC membrane preparations (5 ug aliquots) were electrophoresed on a nonreducing SDS-PAGE (10%) , and then electroblotted onto a nitrocellulose filter. Following blocking for 1 hour in a solution of PBS containing 1.5% BSA, the nitrocellulose filter was probed with ¹²⁵ I- AGE-BSA in the presence of 100-fold excess of either BSA or AGE-BSA. The blot was washed 3 times with IX PBS and exposed to Kodak XAR-5 film at -80°C.

Uptake and degradation: MC uptake and degradation was performed with a minor modification of previously described procedures. Briefly, MCs were grown to confluency in 6-well plates in DMEM containing 20% FBS and insulin. MC accumulation of radioactive ligand

(AGE-BSA) was assessed by incubating cells with various concentrations of ¹²⁵ I-AGE-BSA, in the presence and absence

of 100-fold excess of unlabelled AGE-BSA, for 4 hours at 37°C . After washing the cells 3 times with ice-cold PBS, the cells were solubilized in 1% Triton X-100 for 45 minutes at room temperature, and the amount of cell associated radioactivity was determined. Specific uptake was defined by the same criteria as used for the MC binding studies. Protein concentration was determined by the method of Bradford. Degradation was determined by measuring TCA-soluble radioactivity in the aspirated medium.

Proliferation assays: Rat MCs in DMEM containing 20% FBS were plated at 1X10 ⁴ cells/well onto flat-bottom 96-well microtitre plates, which had been pre-coated with different amounts of either AGE-modified or unmodified matrix proteins. 24 hours later, the cells were washed with IX PBS and incubated for an additional 48 hrs in medium containing 0.3% FBS. The cells were then labelled with 2 uCi of ( ³ H) thymidine (Amersham Corp., Arlington Heights,IL) for 18-24 hours, following which the supernatants were aspirated and the cells in each well were harvested onto glass fiber filters with an automated cell harvester. The amount of ( ³ H) thymidine incorporated was determined with a Beckman Scintillation Counter.

To confirm thymidine incorporation data, parallel studies were carried out using an immunocytochemical assay system for detection of DNA synthesis by measuring bromo- deoxyuridine (BrdU) incorporation, while in separate experiments cells were trypsinized and counted in a

Coulter particle counter. The data obtained by these two additional methods were consistent with ³ H-thymidine results (variations between replicate wells deviated no more than 10%) .

Fibronectin production: Human MCs in RPMI-1640 medium supplemented with 20% FBS were plated at 2 X 10 ⁵ cells per

well onto 6-well plates which had been coated with glucose-modified or unmodified matrix proteins. After 24 hours, the cells were washed with IX PBS and incubated in medium containing 0.3% FBS for 48 hours. The cells were then labeled in methionine free medium for 3 hours with 200 uCi of (35S) methionine and cysteine (Translabel, ICN) .

After labeling, the medium was removed and the cell monolayers were washed with cold IX PBS. The monolayers were extracted with 0.5 ml of a 1M urea solution containing ImM dithiothreitol (DTT) , 10 mM Tris-HCL (pH) 7.4), 10 mM EDTA, and 2 mM PMSF.

Fibronectin was then isolated from the medium and matrix by immunoprecipitation with an IgG purified anti-human fibronectin antibody (Cappel, Malvern, PA) . Anti-fibronectin antibody was added to the samples and incubated overnight at 4°C. To insure than any differences in fibronectin synthesis were not due to different number of cells/well, equal amounts of TCA- precipitable counts were immunoprecipitated from each well. The immune complexes were isolated using protein A-Sepharose beads (Pharmacia) .

After washing the protein A-Sepharose beads 3 times in lOOmM Tris HCl (pH 7.4), 0.5% SDS, 0.5% Triton X-100, 2 mM PMSF, and 10 mM EDTA, fibronectin was released by heating at 100°C for 5 minutes in SDS-PAGE sample buffer, and analyzed by gel electrophoresis and fluorography. The amount of fibronectin from each sample was quantitated by slicing the fibronectin band from the gel and determining (35S) methionine and cysteine incorporation in a liquid scintillation counter.

This invention may be embodied in other forms or carried out in other ways without departing from the spirit or

essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.