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Title:  Methods and composition for detecting and treating kidney diseases associated with adhesion of crystals to kidney cells

United States Patent:  6,043,216

Inventors:  Toback; F. Gary (Chicago, IL); Lieske; John C. (Evanston, IL)

Assignee:  Arch Development Corporation (Chicago, IL)

Appl. No.:  837226

Filed:  April 10, 1997

Abstract

An autocrine crystal adhesion inhibitor called CAI is an anionic, sialic acid-containing glycoprotein secreted by kidney epithelial cells that blocks adhesion of calcium oxalate monohydrate (COM) crystals to the cell surface. Novel amino acid sequences are shown for the amino-acid terminus and 6 interval fragments. Persons may be classified according to risk of developing kidney stones, by measuring the amount of CAI in a biological sample. Treatment efficacy is also monitored by this method. CAI is administered in vivo to prevent nephrolithiasis. A rapid, simple assay to detect agents that inhibit adhesion of COM crystals to the surface of kidney epithelial cells is characterized.

SUMMARY OF THE INVENTION

A composition which disrupts the cascade of events that results in crystal retention and nephrolithiasis is the crystal adhesion inhibitor (CAI) of the present invention, a composition which is a sialic acid-containing anionic glycoprotein having an estimated molecular weight of 39,000 daltons based on SDS polyacrylamide gel electrophoresis. The sialic acid residues are important in maintaining a negative charge, because the amino acid composition of the inhibitor predicts a net near-neutral charge.

Renal epithelial cells constitutively produce the factor (CAI) that prevents adhesion of COM crystals to their apical surface. Sequence information for 124 amino acids of the NH2 -terminus and 6 internal fragments of this novel core protein has been obtained, and used to prepare 2 monospecific antibodies against 2 different synthetic peptides.

The inhibitor is purified by a novel crystal-affinity method wherein the anionic, hydrophobic material adheres to the crystals from which it is later removed by, e.g., EDTA. This is followed by purification using SDS-PAGE and electroblotting or electro elution of the gel or reversed-phase HPLC. The purified CAI is an anionic glycoprotein. The presence of carbohydrate is manifested by a loss of inhibitory activity following exposure of CAI to neuraminidase, indicating the critical functional importance of its anionic sialic acid residues. Its carbohydrate character is confirmed by the detection of uronic acid using the carbazole reaction, and a positive test using a DIG glycan kit. Its protein character is established by amino acid compositional analysis and amino acid sequence information, and is supported by positive reactions in the presence of ninhydrin or bicinchoninic acid (BCA). Its activity is resistant to pH 2, freezing and thawing. The near neutral net charge of the CAI protein distinguishes it from known strongly anionic proteins that block adhesion of COM crystals to the surface of renal epithelial cells.

Availability of the CAI permits comparative screening for other candidate inhibitors of crystal adhesion. Generally, those at least as active in preventing crystal adhesion to cells, are selected for further processing. The tissue culture system of the present invention is contacted with a candidate agent, and the degree of inhibition of crystal adhesion to cells in a control culture are each compared to a culture treated with CAI as a standard.

Assays based on crystal adhesion are useful for identifying patients at high risk for kidney stone disease and for screening for drugs which prevent crystal adhesion. Polyclonal antibodies developed against the CAI by standard methods are used to quantity the amount of CAI in a sample of urine from an individual with untreated or treated nephrolithiasis, or who is suspected of having this condition.

For characterization of the CAI, a monospecific polyclonal anti serum is preferable to monoclonal antibodies because the latter each complex with single antigenic determinants, whereas a polyclonal an tiserum likely recognizes multiple sites on the target molecule. Because CAI is a glycoprotein and neuraminidase treatment inhibits its function, it is likely that sialic acid residues are present at its "active site(s)", the sites by which the CAI binds to crystal surfaces or cell surfaces to block crystal adhesion. Thus a polyclonal anti serum which contains IgG molecules that recognize antigenic determinants composed of carbohydrate, protein, or both is a particularly useful reagent. Whereas a monoclonal antibody might recognize antigenic sites on the CAI molecule which are not important for its biological function, a polyclonal antibody is more likely to block activity when it is recognized and binds to CAI. Monospecific antibodies have been prepared to two synthetic peptides having sequences identified in CAI. Monoclonal antibodies can also be prepared to functionally active fragments of the CAI used as immunogen employing techniques well-known to the art.

To determine the minimum amount of the molecule necessary to elicit biological activity, that is, the minimum peptide that includes the "active site or sites," CAI that has been isolated and purified as described herein is subjected to enzymatic cleavage which produces fragments. The fragments are then tested for biological activity according to the methods described herein. It is likely that the active site or sites will include sialic acid residues. The relative efficiency of the active-site containing fragments is also of interest because even though biologically active, some fragments are likely to be more active than others, e.g. a heparin molecule of 6,000 daltons molecular weight is not as efficient an adhesion inhibitor as a heparin molecule of 18,000 daltons molecular weight. Inhibitors are designed to complex with the active site(s) as determined by the methods discussed above.

Susceptibility to stone formation varies among individuals. This variation is likely due to inherent variations among individuals in the ability to produce inhibitor, which is measurable and provides a means for classifying persons according to risk of developing stones. If an immunological assay detects no CAI or an amount of CAI less than the value in non affected control individuals, the patient is considered to be at increased risk of kidney stone formation. This assay is also useful for monitoring the success of therapeutic regimens designed to treat or prevent the appearance of new stones which may be directly correlated to the urinary concentration of CAI in specific individuals. The amount of CAI detected by the immunological assay and its functional capacity to inhibit adhesion of COM crystals to kidney epithelial cells in culture is used to classify patients with nephrolithiasis. A tissue culture system containing kidney epithelial cells is used to quantity the function of the CAI or its equivalent.

Novel molecular tools are now available to seek the cDNA sequence of CAI. Amino acid sequence information now available (124 residues) permits studies using a polymerase chain reaction (PCR) strategy, whereas the two monospecific antisera are useful in an immunoscreening approach to obtain a cDNA sequence.

After the DNA sequence of the gene encoding CAI is obtained, further study of the protein and the factors that regulate its production will improve understanding of kidney stone formation. In certain individuals, defective production of CAI likely predisposes to kidney stone formation. In CAI-deficient patients, adhesion of crystals to tubular cells is expected to occur more readily, and once retained in the nephron, such crystals likely grow into kidney stones. Therefore, study of CAI could yield important new insights into the pathogenesis of kidney stone formation, and lead to development of a novel form of therapy by using knowledge about CAI structure and function.

To summarize uses of CAI:

1. Urine from a patient is tested to detect defective CAI or other anti-adhesion factors in urine by using the crystal adhesion assay described herein.

2. Diverse chemical and pharmaceutical agents are selected because they exhibit characteristics similar to CAI, and are tested for their ability to prevent crystal adhesion to cells as compared to CAI.

3. The structure of CAI provides a basis for the rational design of effective drugs, for example, an active domain of the CAI molecule that is smaller than the native molecule in size may be used to prevent crystal adhesion.

4. CAI is used as a drug to prevent stones: delivery is via liposome, intravenous or subcutaneous injection, or intranasal systems. Chemical modification of CAI may permit its use by an oral route.

5. Monospecific antibodies to purified CAI or peptide fragments of CAI are provided for detection and quantitative assays of CAI.

6. Hybridization of CAI cDNA with samples of DNA from individuals is used to screen for defective CAI carriers who are candidates for stone disease. Sloughed renal tubular cells isolated from urine or white blood cells from peripheral blood are suitable samples for this hybridization test.

7. If a crystal-binding receptor is identified on a cell surface, sloughed renal tubular cells could be screened for an abnormal quantity/quality of the receptor.

Claim 1 of 5 Claims

1. A purified inhibitor of adhesion of calcium oxalate monohydrate (COM) crystals to kidney epithelial cells, said crystal adhesion inhibitor (CAI) having the following characteristics:

(a) an estimated molecular weight of 39,000 daltons based on SDS-polyacrylamide gel electrophoresis;

(b) an anionic glycoprotein containing sialic acid; and

(c) an amino acid sequence selected from the group consisting of the seven sequences as follows:

1) (K/D)INGGGATLPQPLYQTSGVLTAGFAP YI (S) V (N) (A) (K) SEQ.ID. NO: 4;

2) (F) VVTTNFADSFGVPAGAVPAVTSQGVM D (S/A) (L) (N)SEQ.ID. NO: 5;

3) LIQVPSVATSVAIPFNK SEQ.ID. NO: 6;

4) LAFLNNDYSQFGTGTK SEQ.ID. NO: 7;

5) AAITSNFVTATSAL SEQ.ID. NO: 8;

6) LTSTELSTYASTK SEQ.ID. NO: 9;

7) NRFVPLPTAX(K) SEQ.ID. NO: 10.


 

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