WO2013070741A1 - Methods and materials for regulating natriuretic polypeptide function - Google Patents

Abstract

This document provides methods and materials for using natriuretic polypeptides. For example, methods and materials related to identifying mammals having the ability to respond to treatment with a natriuretic polypeptide as well as methods and materials for increasing a mammal's ability to respond to treatment with a natriuretic polypeptide are provided.

Description

METHODS AND MATERIALS FOR REGULATING NATRIURETIC

POLYPEPTIDE FUNCTION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 61/558,653, filed November 11, 2011. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

BACKGROUND

. Technical Field

This document relates to methods and materials for using natriuretic polypeptides. For example, this document provides methods and materials related to identifying mammals having the ability to respond to treatment with a natriuretic polypeptide as well as methods and materials for increasing a mammal's ability to respond to treatment with a natriuretic polypeptide.

2. Background Information

Natriuretic polypeptides are polypeptides that can cause natriuresis (increased sodium excretion in the urine). Such polypeptides can be produced by brain, heart, kidney, and/or vascular tissue. The natriuretic peptide family in humans includes the cardiac hormones atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C- type natriuretic peptide (CNP), and urodilatin (URO). Natriuretic polypeptides function via guanylyl cyclase receptors (i.e., NPR-A for ANP, BNP, and URO; and NPR-B for CNP) and the second messenger cyclic 3 '5' guanosine monophosphate (cGMP) (Kuhn, Circ. Res., 93:700-709 (2003); Tawaragi et al., Biochem. Biophys. Res. Commun., 175:645-651 (1991); and Komatsu et al., Endocrinol., 129: 1104-1106 (1991)).

SUMMARY

This document provides methods and materials for using natriuretic polypeptides. For example, this document provides methods and materials for identifying mammals having the ability to respond to treatment with a natriuretic polypeptide or response to endogenous levels of natriuretic polypeptide. As described herein, mammals (e.g., humans) can be assessed for normal or elevated levels of protein disulfide isomerase activity on cell surfaces (e.g., cell surfaces of circulating blood cells or cells from tissues). Those mammals having normal or elevated levels of PDI polypeptide activity on cell surfaces can be identified as being capable of responding to a treatment with a natriuretic polypeptide. Those mammals having a reduced level or undetectable level of PDI polypeptide activity on cell surfaces can be identified as having a reduced capability of responding to a treatment with a natriuretic polypeptide.

Having the ability to identify mammals capable of responding to treatment with a natriuretic polypeptide can allow physicians to select proper natriuretic polypeptide treatments for particular patients in an efficient manner. In addition, having the ability to identify mammals have a reduced capability of responding to treatment with a natriuretic polypeptide can allow physicians to select non-natriuretic polypeptide treatments or combination treatments designed to increase natriuretic polypeptide responsiveness for particular patients in an efficient manner. For example, a mammal having a

cardiovascular and/or renal condition and identified as having a reduced capability of responding to treatment with a natriuretic polypeptide can be treated with vasodilators (e.g., nitroglycerin) or ionotropic medications or a combination of one or more natriuretic polypeptides and one or more agents designed to increase natriuretic polypeptide responsiveness (e.g., soluble PDI polypeptides, or RNA- or DNA-based delivery agents capable of expressing PDI polypeptides).

This document also provides methods and materials for increasing a mammal's ability to respond to treatment with a natriuretic polypeptide. For example, one or more agents designed to increase natriuretic polypeptide responsiveness can be administered to a mammal under conditions wherein the mammal's ability to respond to treatment with a natriuretic polypeptide is increased. In some cases, soluble PDI polypeptides or RNA- or DNA-based delivery agents capable of expressing PDI polypeptides can be administered to a mammal under conditions wherein the mammal's ability to respond to treatment with a natriuretic polypeptide is increased. As described herein, the presence of a normal or elevated level of PDI polypeptide activity can allow cells to respond to treatment with a natriuretic polypeptide, while inhibition of PDI polypeptide activity reduces the ability of cells to respond to treatment with a natriuretic polypeptide. Having the ability to increase PDI polypeptide activity and thereby increase a mammal's ability to respond to treatment with a natriuretic polypeptide can allow physicians to treat patients having a

cardiovascular and/or renal condition with a natriuretic polypeptide in an effective manner that would not have been as effective without increasing PDI polypeptide activity.

In general, one aspect of this document features a method for identifying a mammal unlikely to respond to treatment with a natriuretic polypeptide. The method comprises, or consists essentially of, (a) detecting the presence of a reduced level of cell surface or circulating PDI polypeptide activity of the mammal, and (b) classifying the mammal as being unlikely to respond to the treatment based at least in part on the presence. The mammal can be a human. The reduced level can be an undetectable level.

In another aspect, this document features a method for identifying a mammal likely to respond to treatment with a natriuretic polypeptide. The method comprises, or consists essentially of, (a) detecting the presence of a normal or elevated level of cell surface or circulating PDI polypeptide activity of the mammal, and (b) classifying the mammal as being likely to respond to the treatment based at least in part on the presence. The mammal can be a human. The method can comprise detecting the presence of an elevated level of cell surface or circulating PDI polypeptide activity of the mammal. The method can comprise detecting the presence of a normal level of cell surface or circulating PDI polypeptide activity of the mammal.

In another aspect, this document features a method for treating a mammal having a reduced level of endogenous natriuretic polypeptide responsiveness. The method comprises, or consists essentially of, (a) administering, to the mammal, an agent under conditions wherein the level of PDI activity is increased, and (b) administering, to the mammal, a natriuretic polypeptide. The mammal can be a human. The agent can be a PDI polypeptide. The natriuretic polypeptide can be ANP, BNP, CNP, ASBNP,

ANX042, or a chimeric natriuretic peptide. The chimeric natriuretic peptide can be CD- NP. In another aspect, this document features a method for treating a mammal. The method comprises, or consists essentially of, (a) detecting the presence of a reduced level of cell surface or circulating PDI polypeptide activity of the mammal, (b) administering, to the mammal, an agent under conditions wherein the level of PDI activity is increased, and (c) administering, to the mammal, a natriuretic polypeptide. The mammal can be a human. The agent can be a PDI polypeptide. The natriuretic polypeptide can be ANP, BNP, CNP, ASBNP, ANX042, or a chimeric natriuretic peptide. The chimeric natriuretic peptide can be CD-NP.

In another aspect, this document features a method for treating a mammal. The method comprises, or consists essentially of, (a) detecting the presence of a normal or elevated level of cell surface or circulating PDI polypeptide activity of the mammal, and (b) administering, to the mammal, a natriuretic polypeptide. The mammal can be a human. The natriuretic polypeptide can be ANP, BNP, CNP, ASBNP, ANX042, or a chimeric natriuretic peptide. The chimeric natriuretic peptide can be CD-NP.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

Figure 1 A is a photograph of a ID Far western blot. Membrane proteins from human mesangial cells were separated ID SDS-PAGE. Proteins were transferred and labeled with a truncated ASBNP polypeptide (ANX-042). Following washing, immunodetection of ANX-042 was performed with an antibody to the BNP ring, which is detects ANX-042. Binding was seen at approximately 60 kD and 115 kD. Figure IB is a photograph of a 2D Far western blot. Membrane proteins from human mesangial cells were separated on 2D gels. Proteins were transferred and labeled with ANX-042.

Following washing, immunodetection of ANX-042 was performed with an antibody to the BNP ring, which detects ANX-042. Five immuno-reactive spots were isolated and sequenced by mass spectometry. Sequences from a PDI polypeptide (P4HB) were identified in spots M2, M3, and M5.

Figure 2A is a photograph of immunodetection results of cellular proteins that identified PDI expressed in vascular smooth muscle cells (VSMCs), endothelial cells (HUVECs) and mesangial cells (HMCs). Expression of PDI was not detected on LLC- PKl cells (porcine renal tubular cells). Figure 2B demonstrates that PDI and NPR-B co- localize in HMC lysates. Proteins were pulled down with an anti-NPRB antibody (Santa- Cruz) and detected with an anti-PDI antibody (RL90, Novus) and compared with non- immunoprecipated proteins from HMCs.

Figure 3 A contains immunocytochemistry data demonstrating that PDI and NPR- B are co-expressed and co-localize in human mesangial cells. Figure 3B contains immunocytochemistry data demonstrating that expression of PDI and NPRB is not detected in LLC-PKl cells.

Figure 4 contains graphs plotting the amount of cGMP (fmol/well) generated in

HMCs (A), HUVECS (B), and HSMCS (C), and LLC-PKl cells (D) treated as indicated.

Figure 5 contains results demonstrating the inhibition of cGMP stimulation by an anti-PDI antibody (RL90).

Figures 6A and 6B contain results demonstrating that siRNA-mediated knockdown of PDI attenuates CNP -mediated cGMP activation in human mesangial cells.

Figure 7 is a graph plotting results indicating that addition of recombinant PDI (Sigma) enhances cGMP production in LLC-PKl cells, which are cells that do not express PDI.

Figure 8 is a listing of a nucleic acid sequence (SEQ ID NO: l) encoding a human PDI polypeptide. Figure 9 is a listing of an amino acid sequence (SEQ ID NO:2) of a human PDI polypeptide.

DETAILED DESCRIPTION

This document provides methods and materials for using natriuretic polypeptides.

For example, this document provides methods and materials for identifying mammals having the ability to respond to treatment with a natriuretic polypeptide. As described herein, mammals can be assessed for normal, elevated, or reduced levels of PDI polypeptide activity on cell surfaces. A mammal identified as having normal or elevated levels of PDI polypeptide activity on cell surfaces can be classified as being capable of responding to a treatment with a natriuretic polypeptide, while a mammal identified as having a reduced level or undetectable level of PDI polypeptide activity on cell surfaces can be classified as having a reduced capability of responding to a treatment with a natriuretic polypeptide.

The term "natriuretic polypeptide" as used herein refers to any polypeptide having diuretic or natriuretic activity. Examples of natriuretic polypeptides include, without limitation, human ANP, human BNP, human CNP, human URO, Dendroaspis natriuretic peptide (DNP), alternatively spliced BNP (ASBNP; see PCT/US2008/076434), and CD- NP (see, e.g., Lee et al, J. Clin. Pharmacol, 49(6):668-673 (2009)).

Any appropriate mammal can be assessed for a normal, elevated, or reduced level of PDI polypeptide activity on cell surfaces. For example, humans, non-human primates, monkeys, horses, cows, sheep, pigs, dogs, and cats can be assessed for a normal, elevated, or reduced level of PDI polypeptide activity on cell surfaces. In some cases, a human diagnosed as having a cardiovascular and/or renal condition and being in need of treatment with a natriuretic polypeptide can be assessed for a normal, elevated, or reduced level of PDI polypeptide activity on cell surfaces to determine whether or not the human is likely to respond to the natriuretic polypeptide treatment.

When assessing a mammal for the level of PDI polypeptide activity on cell surfaces, any appropriate cells can be evaluated. For example, circulating blood cells or cells obtained from tissue can be obtained from a mammal and tested to determine the level of cell surface PDI polypeptide activity. In some cases, the level of PDI polypeptide expression on cell surfaces can be measured to determine whether a mammal has a normal, elevated, or reduced level of PDI polypeptide activity on cell surfaces. In some cases, cells within a blood sample can be assessed to determine the level of cell surface PDI polypeptide expression per mL of blood. Any appropriate method can be used to assess the level of cell surface PDI polypeptide expression. For example, immunological assays such as Western blots, FACS analyses, ELISAs, and RIAs can be used to determine whether or not a mammal has normal, elevated, or reduced levels of PDI polypeptide expression on cell surfaces.

In immunological assays, an antibody having specific binding affinity for a PDI polypeptide provided herein or a secondary antibody that binds to such an antibody can be labeled, either directly or indirectly. Suitable labels include, without limitation, radionuclides (e.g., ¹²⁵I, ¹³¹1, ³⁵S, ³H, ³²P, ³³P, or ¹⁴C), fluorescent moieties (e.g., fluorescein, FITC, PerCP, rhodamine, or PE), luminescent moieties (e.g., Qdot™ nanoparticles supplied by Invitrogen (Carlsbad, CA)), compounds that absorb light of a defined wavelength, or enzymes (e.g., alkaline phosphatase or horseradish peroxidase). Antibodies can be indirectly labeled by conjugation with biotin then detected with avidin or streptavidin labeled with a molecule described above. Methods of detecting or quantifying a label depend on the nature of the label and are known in the art. Examples of detectors include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and

densitometers. Combinations of these approaches (including "multi-layer" assays) familiar to those in the art can be used to enhance the sensitivity of assays.

Immunological assays for detecting a PDI polypeptide can be performed in a variety of formats including, without limitation, sandwich assays, competition assays (competitive RIA), or bridge immunoassays. See, for example, U.S. Patent Nos.

5,296,347; 4,233,402; 4,098,876; and 4,034,074. Methods of detecting a PDI

polypeptide generally include contacting a biological sample (e.g., cells or a cell membrane preparation) with an antibody that binds to a PDI polypeptide and detecting binding of the PDI polypeptide to the antibody. For example, an antibody having specific binding affinity for a human PDI polypeptide having the amino acid sequence set forth in SEQ ID NO:2 can be immobilized on a solid substrate by any of a variety of methods known in the art and then exposed to the biological sample. Binding of the PDI polypeptide to the antibody on the solid substrate can be detected by exploiting the phenomenon of surface plasmon resonance, which results in a change in the intensity of surface plasmon resonance upon binding that can be detected qualitatively or

quantitatively by an appropriate instrument, e.g., a Biacore apparatus (Biacore

International AB, Rapsgatan, Sweden). In some cases, the antibody can be labeled and detected as described above. A standard curve using known quantities of a PDI polypeptide can be generated to aid in the quantitation of the levels of the PDI polypeptide.

In some cases, cell sorting such as a FACS analysis can be performed to determine the level of cell surface PDI polypeptide expression.

In some embodiments, a "sandwich" assay in which a capture antibody is immobilized on a solid substrate can be used to detect the expression level of a PDI polypeptide. The solid substrate can be contacted with a biological sample (e.g., a polypeptide preparation from cell membranes) such that any PDI polypeptide in the sample can bind to the immobilized antibody. The presence, absence, or level of PDI polypeptide bound to the antibody can be determined using a "detection" antibody having specific binding affinity for the PDI polypeptide. It is understood that in sandwich assays, the capture antibody should not bind to the same epitope (or range of epitopes in the case of a polyclonal antibody) as the detection antibody. Thus, if a monoclonal antibody is used as a capture antibody, the detection antibody can be another monoclonal antibody that binds to an epitope that is either physically separated from or only partially overlaps with the epitope to which the capture monoclonal antibody binds, or a polyclonal antibody that binds to epitopes other than or in addition to that to which the capture monoclonal antibody binds. If a polyclonal antibody is used as a capture antibody, the detection antibody can be either a monoclonal antibody that binds to an epitope that is either physically separated from or partially overlaps with any of the epitopes to which the capture polyclonal antibody binds, or a polyclonal antibody that binds to epitopes other than or in addition to that to which the capture polyclonal antibody binds. Sandwich assays can be performed as sandwich ELISA assays, sandwich Western blotting assays, or sandwich immunomagnetic detection assays. Suitable solid substrates to which an antibody (e.g., a capture antibody) can be bound include, without limitation, microtiter plates, tubes, membranes such as nylon or nitrocellulose membranes, and beads or particles (e.g., agarose, cellulose, glass, polystyrene, polyacrylamide, magnetic, or magnetizable beads or particles). Magnetic or magnetizable particles can be particularly useful when an automated immunoassay system is used.

Antibodies having specific binding affinity for a PDI polypeptide can be produced through standard methods. For example, a PDI polypeptide can be recombinantly produced or can be chemically synthesized, and used to immunize host animals, including rabbits, chickens, mice, guinea pigs, or rats. For example, a PDI polypeptide having the amino acid sequence set forth in SEQ ID NO:2 can be used to immunize an animal. Various adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Monoclonal antibodies can be prepared using a PDI polypeptide and standard hybridoma technology. In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described by Kohler et al., Nature, 256:495 (1975), the human B- cell hybridoma technique (Kosbor et al., Immunology Today, 4:72 (1983); Cole et al.,

Proc. Natl. Acad. Sci. USA, 80:2026 (1983)), and the EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc., pp. 77-96 (1983)). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridoma producing the monoclonal antibodies can be cultivated in vitro and in vivo.

In some cases, anti-PDI polypeptide antibodies can be obtained commercially. For example, anti-PDI polypeptide antibodies can be obtained from Novus Biologies (e.g., RL90).

Other techniques for assessing the level of cell surface PDI polypeptide activity or expression include biological assays such as disulfide isomerase activity assays.

Examples of such disulfide isomerase activity assays are described elsewhere (Raturi and Mutus, Free Radic. Biol. Med., 43:62-70 (2007); Xiao and Gordge, Nitric Oxide, 25(3):303-8 (2011); and Holmgren, J. Biol. Chem., 254:9627-9632 (1979)).

Once the level of cell surface PDI polypeptide expression or activity is measured, it can be assessed to determine if the level of PDI polypeptide activity is a normal, elevated, or reduced level. The term "elevated" as used herein with respect to the level of cell surface PDI polypeptide activity refers to any level that is greater than a control level of cell surface PDI polypeptide activity from normal, healthy mammals known to respond to natriuretic polypeptides. The term "normal" as used herein with respect to the level of cell surface PDI polypeptide activity refers to any level that is similar to a control level of cell surface PDI polypeptide activity from normal, healthy mammals known to respond to natriuretic polypeptides. The term "reduced" as used herein with respect to the level of cell surface PDI polypeptide activity refers to any level that is less than a control level of cell surface PDI polypeptide activity from normal, healthy mammals known to respond to natriuretic polypeptides. In some cases, a reduced level of cell surface PDI polypeptide activity can be an undetectable level of cell surface PDI polypeptide activity or expression. In some cases, a reduced level of cell surface PDI polypeptide activity can be any level that is less than a reference level for a reduced level of cell surface PDI polypeptide activity or expression. For example, a reference level of cell surface PDI polypeptide activity can be the average level of cell surface PDI polypeptide expression that is present in samples obtained from a random sampling of 50 healthy mammals (e.g., humans) known to respond to natriuretic polypeptides. It will be appreciated that levels from comparable samples are used when determining whether or not a particular level is a normal, elevated, or reduced level.

In some cases, a nucleic acid-based assay can be used to determine whether a mammal contains a normal, elevated, or reduced level of PDI polypeptide expression on cell surfaces. For example, nucleic acid encoding a PDI polypeptide or controlling the expression of a PDI polypeptide can be assessed for the presence of a genetic mutation or alteration that increases or decreases expression of a functional PDI polypeptide. In some cases, the presence of a truncation mutation, frame-shift mutation, or deletion can indicate that the mammal contains a reduced level of PDI polypeptide expression or activity on cell surfaces. Any appropriate method can be used to detect such genetic mutations or alterations. For example, sequencing techniques, SNP detection assays, PCR-based assays, and high throughput DNA sequencing can be used to assess nucleic acid encoding a PDI polypeptide or controlling the expression of a PDI polypeptide for the presence or absence of genetic mutations or alterations that increase or decrease expression of a functional PDI polypeptide.

A mammal determined to have a normal or elevated level of PDI polypeptide activity on cell surfaces can be classified as being capable of responding to a treatment with a natriuretic polypeptide. For example, a human having a normal level of cell surface PDI polypeptide activity can be classified as being capable of responding to a treatment with a natriuretic polypeptide. Such a human, when in need of treatment with a natriuretic polypeptide, can be treated with one or more natriuretic polypeptides without requiring any additional treatments designed to increase responsiveness to a natriuretic polypeptide.

A mammal determined to have a reduced level or undetectable level of PDI polypeptide activity on cell surfaces can be classified as having a reduced capability of responding to a treatment with a natriuretic polypeptide. For example, a human having a reduced level of cell surface PDI polypeptide activity or an undetectable level of cell surface PDI polypeptide expression can be classified as having a reduced capability of responding to a treatment with a natriuretic polypeptide. Such a human, when in need of treatment with a natriuretic polypeptide, can be treated with either a treatment option that is an alternative to treatment with a natriuretic polypeptide or a treatment option that includes a natriuretic polypeptide in combination with an agent designed to increase responsiveness to a natriuretic polypeptide. Examples of agents designed to increase responsiveness to a natriuretic polypeptide that can be used in combination with one or more natriuretic polypeptides include, without limitation, soluble PDI polypeptides or DNA- or RNA-based delivery agents capable of expressing a PDI polypeptide.

In general, a gene delivery agent capable of expressing a PDI polypeptide can be a nucleic acid construct or expression vector designed to be delivered to cells and express a PDI polypeptide. As used herein, the term "nucleic acid" refers to both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. A nucleic acid molecule can be double-stranded or single-stranded (i.e., a sense or an antisense single strand). Nucleic acids include, for example, cDNAs encoding a PDI polypeptide such as a PDI polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

An "isolated nucleic acid" is a nucleic acid that is separated from other nucleic acid molecules that are present in a vertebrate genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a vertebrate genome. The term "isolated" as used herein with respect to nucleic acids also includes any non-naturally- occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally- occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a DNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA libraries or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not considered an isolated nucleic acid.

Isolated nucleic acid molecules can be produced using standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence that encodes a PDI polypeptide. PCR refers to a procedure or technique in which target nucleic acids are enzymatically amplified. Sequence information from the ends of the region of interest or beyond typically is employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995. When using RNA as a source of template, reverse transcriptase can be used to synthesize

complementary DNA (cDNA) strands. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication, or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis, Genetic Engineering News, 12: 1 (1992); Guatelli et al., Proc. Natl. Acad. Sci. USA 87: 1874-1878 (1990); and Weiss, Science, 254: 1292 (1991).

Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., > 100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of

complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the

oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.

Sources of nucleotide sequences from which nucleic acid molecules encoding a PDI polypeptide, or the nucleic acid complement thereof, can be obtained include total or polyA⁺ RNA from any mammalian (e.g., human, rat, mouse, canine, bovine, equine, ovine, caprine, or feline) cellular source from which cDNAs can be derived by methods known in the art. Other sources of the nucleic acid molecules include genomic libraries derived from any mammalian source.

Nucleic acid molecules encoding a PDI polypeptide can be identified and isolated using standard methods, e.g., as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989). For example, reverse-transcriptase PCR (RT-PCR) can be used to isolate and clone a PDI cDNA from isolated RNA that contains RNA sequences of interest (e.g., total RNA isolated from human tissue). Other approaches to identify, isolate, and clone PDI cDNAs include, for example, screening cDNA libraries.

Vectors containing nucleic acids such as those described herein also are provided. A "vector" is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. An "expression vector" is a vector that includes one or more expression control sequences, and an "expression control sequence" is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.

In the expression vectors, a nucleic acid (e.g., a nucleic acid encoding a PDI polypeptide) can be operably linked to one or more expression control sequences. As used herein, "operably linked" means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 to 500 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter.

Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the

transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is "operably linked" and "under the control" of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.

Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno- associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA).

An expression vector can include a tag sequence designed to facilitate subsequent manipulation or detection of the expressed nucleic acid sequence. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.

In some cases, after determining that a mammal suffering from acute heart failure, acute myocardial infarction, renal failure, liver failure, or other volume overloaded states is likely to respond to a natriuretic polypeptide treatment as described herein, that mammal can be treated with one or more natriuretic polypeptides. For example, a natriuretic polypeptide can be administered to a mammal (e.g., a human or a non-human mammal) in order to reduce or inhibit cardiac remodeling that can occur, for example, after myocardial infarction. In some embodiments, for example, a natriuretic polypeptide can be administered to a mammal diagnosed as having had an AMI. The natriuretic polypeptide or composition containing a natriuretic polypeptide can be administered at any suitable dose, depending on various factors including, without limitation, the agent chosen, the disease, and whether prevention or treatment is to be achieved.

Administration can be local or systemic.

In some embodiments, a natriuretic polypeptide can be administered at a dose of at least about 0.01 ng polypeptide/kg to about 100 mg polypeptide/kg of body mass (e.g., about 10 ng polypeptide/kg to about 50 mg polypeptide/kg, about 20 ng polypeptide/kg to about 10 mg polypeptide/kg, about 0.1 ng polypeptide/kg to about 20 ng polypeptide/kg, about 3 ng polypeptide/kg to about 10 ng polypeptide/kg, or about 50 ng polypeptide/kg to about 100 μg/kg) of body mass, although other dosages also may provide beneficial results. In some cases, a natriuretic polypeptide can be administered as a continuous intravenous infusion beginning at or about the time of reperfusion (i.e., at the time the occluded artery is opened), and continuing for one to seven days (e.g., one, two, three, four, five, six, or seven days). Such a natriuretic polypeptide can be administered at a dose of, for example, about 0.1 ng polypeptide/kg/minute to about 500 ng polypeptide/kg/minute (e.g., about 0.5 ng polypeptide/kg/minute, about 1 ng

polypeptide/kg/minute, about 2 ng polypeptide/kg/minute, about 3 ng

polypeptide/kg/minute, about 5 ng polypeptide/kg/minute, about 7.5 ng

polypeptide/kg/minute, about 10 ng polypeptide/kg/minute, about 12.5 ng

polypeptide/kg/minute, about 15 ng polypeptide/kg/minute, about 20 ng

polypeptide/kg/minute, about 25 ng polypeptide/kg/minute, about 30 ng

polypeptide/kg/minute, about 50 ng polypeptide/kg/minute, about 100 ng

polypeptide/kg/minute, or about 300 ng polypeptide/kg/minute). In some embodiments, a natriuretic polypeptide can be administered before reperfusion (e.g., about one hour prior to reperfusion), either as one or more individual doses or as a continuous infusion beginning about one hour prior to reperfusion). For example, a natriuretic polypeptide can be administered beginning about one hour, about 45 minutes, about 30 minutes, or about 15 minutes prior to reperfusion. In some cases, a natriuretic polypeptide can be administered after reperfusion (e.g., within about ten hours of reperfusion), and can be administered either as one or more individual doses or as a continuous infusion beginning within about ten hours of reperfusion. For example, a natriuretic polypeptide can be administered about one hour, about two hours, about three hours, about four hours, about five hours, about six hours, about seven hours, about eight hours, about nine hours, or about ten hours after reperfusion.

In some embodiments, a natriuretic polypeptide can be administered via a first route (e.g., intravenously) for a first period of time, and then can be administered via another route (e.g., topically or subcutaneously) for a second period of time. For example, a natriuretic polypeptide can be intravenously administered to a mammal (e.g., a human) at a dose of about 0.1 ng polypeptide/kg/minute to about 300 ng

polypeptide/kg/minute (e.g., about 1 ng polypeptide/kg/minute to about 15 ng

polypeptide/kg/minute, about 3 ng polypeptide/kg/minute to about 10 ng

polypeptide/kg/minute, or about 10 ng polypeptide/kg/minute to about 30 ng

polypeptide/kg/minute) for one to seven days (e.g., one, two, three, four, five, six, or seven days), and subsequently can be subcutaneously administered to the mammal at a dose of about 10 ng polypeptide/kg/day to about 100 ng polypeptide/kg/day (e.g., about 10 ng polypeptide/kg/day, about 20 ng polypeptide/kg/day, about 25 ng polypeptide/kg/day, about 30 ng polypeptide/kg/day, about 50 ng polypeptide/kg/day, or about 100 ng polypeptide/kg/day) for five to 30 days (e.g., seven, 10, 14, 18, 21, 24, or 27 days).

The methods provided herein can include administering to a mammal an effective amount of a natriuretic polypeptide. As used herein, the term "effective amount" is an amount of a molecule or composition that is sufficient to alter one or more (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) parameters indicative of reduced cardiac remodeling and/or kidney protection in a mammalian recipient by at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%). For example, an effective amount of a natriuretic polypeptide is an amount that can increase ejection fraction, glomerular filtration rate (GFR), urinary sodium excretion (UNaV), or urine flow (UV) by at least 10%, and/or that can decrease plasma rennin activity (PRA), left ventricular (LV) mass, pulmonary capillary wedge pressure (PCWP), right atrial pressure (RAP), mean aortic pressure (MAP), aldosterone levels, LV hypertrophy, ventricular fibrosis, LV end systolic diameter, and/or fractional reabsorption of sodium by at least 10%, and/or that can result in cardiac unloading. In some embodiments, a method can include administering to a mammal an amount of a natriuretic polypeptide that is sufficient to alter one or more parameters indicative of reduced cardiac remodeling and/or kidney protection by at least 50%>.

In some embodiments, for example, an "effective amount" of a natriuretic polypeptide can be an amount that reduces PRA and MAP and increases GFR and UV in a treated mammal by at least 10% as compared to the levels of those parameters in the mammal prior to administration of the natriuretic polypeptide or without administration of the natriuretic polypeptide (e.g., the level of the parameters observed in a previous myocardial infarction episode).

The invention will be further described in the following example, which does not limit the scope of the invention described in the claims. EXAMPLE

Example 1 - PDI activity enhances cGMP generation by natriuretic peptides

To detect the interaction between natriuretic peptides and receptors on human mesangial cells (HMC) and porcine kidney epithelial cells (LLC-PK1), ID far Western blotting was performed using cell membrane preparations as prey protein and truncated ASBNP (ANX042; SPKMVQGSGCFGRKMDRISSSSGLGCKGKHPLPPRPPSPIPV (SEQ ID NO:3)) as bait protein. An interactive band around 60 kd was identified (Figure 1). Further, 2D far Western blotting was performed, and binding spots were identified and analyzed by mass spectrometry. PDI (P4HB) was identified as a peptide binding partner having a size of ~60kd. Western blotting, immunostaining, and

immunoprecipitation analyses were performed to confirm PDI expression by these cells and to confirm co-localized expression of PDI polypeptides with NPR-B (Figures 2 and 3). To study the roles of PDI on natriuretic peptide stimulation of cGMP, bacitracin, which is a PDI inhibitor (Figure 4), a PDI specific monoclonal antibody RL90 (Figure 5), PDI siRNA (Figure 6), and purified PDI (Figure 7) were used in cGMP analyses.

In human mesangial cells, cGMP generation following ANP, CNP, and ANX042 treatment was reduced by 83%, 80.1%, and 82.6%, respectively. In LLC-PK1 cells, the reductions of cGMP generation following ANP and ANX042 treatment were 28% and 36.6%), respectively. These data suggest that PDI is involved in natriuretic peptide stimulation of cGMP and that the impact of PDI varies among cell types.

Taken together, the results provided herein demonstrate that PDI is a

multifunction cytoplasmic and membrane bound enzyme with additional chaperone activity. It has dithiol-disulfide oxidoreductase activities, which can reduce, oxidize, and isomerize disulfide bonds. As described herein, PDI is capable of acting as a mediator in the interaction of natriuretic peptides and their receptors to regulate cGMP generation.

As described herein, PDI co-localizes with natriuretic peptide receptors, PDI expression varies among cells responsive to natriuretic peptides, and PDI expression, although not necessary for natriuretic peptide activity, appears to regulate cGMP response to natriuretic peptides. OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for identifying a mammal unlikely to respond to treatment with a natriuretic polypeptide, wherein said method comprises:

(a) detecting the presence of a reduced level of cell surface or circulating PDI polypeptide activity of said mammal, and

(b) classifying said mammal as being unlikely to respond to said treatment based at least in part on said presence.

2. The method of claim 1, wherein said mammal is a human.

3. The method of claim 1, wherein said reduced level is an undetectable level.

4. A method for identifying a mammal likely to respond to treatment with a natriuretic polypeptide, wherein said method comprises:

(a) detecting the presence of a normal or elevated level of cell surface or circulating PDI polypeptide activity of said mammal, and

(b) classifying said mammal as being likely to respond to said treatment based at least in part on said presence.

5. The method of claim 4, wherein said mammal is a human.

6. The method of claim 4, wherein said method comprises detecting the presence of an elevated level of cell surface or circulating PDI polypeptide activity of said mammal.

7. The method of claim 4, wherein said method comprises detecting the presence of a normal level of cell surface or circulating PDI polypeptide activity of said mammal.

8. A method for treating a mammal having a reduced level of endogenous natriuretic polypeptide responsiveness, wherein said method comprises: (a) administering, to said mammal, an agent under conditions wherein the level of PDI activity is increased, and

(b) administering, to said mammal, a natriuretic polypeptide.

9. The method of claim 8, wherein said mammal is a human.

10. The method of claim 8, wherein said agent is a PDI polypeptide.

11. The method of claim 8, wherein said natriuretic polypeptide is ANP, BNP, CNP, ASBNP, ANX042, or a chimeric natriuretic peptide.

12. The method of claim 8, wherein said chimeric natriuretic peptide is CD-NP.

13. A method for treating a mammal, wherein said method comprises:

(a) detecting the presence of a reduced level of cell surface or circulating PDI polypeptide activity of said mammal,

(b) administering, to said mammal, an agent under conditions wherein the level of PDI activity is increased, and

(c) administering, to said mammal, a natriuretic polypeptide.

14. The method of claim 13, wherein said mammal is a human.

15. The method of claim 13, wherein said agent is a PDI polypeptide.

16. The method of claim 13, wherein said natriuretic polypeptide is ANP, BNP, CNP, ASBNP, ANX042, or a chimeric natriuretic peptide.

17. The method of claim 13, wherein said chimeric natriuretic peptide is CD-NP.

18. A method for treating a mammal, wherein said method comprises: (a) detecting the presence of a normal or elevated level of cell surface or circulating PDI polypeptide activity of said mammal, and

(b) administering, to said mammal, a natriuretic polypeptide.

19. The method of claim 13, wherein said mammal is a human.

20. The method of claim 13, wherein said natriuretic polypeptide is ANP, BNP, CNP, ASBNP, ANX042, or a chimeric natriuretic peptide.

21. The method of claim 13, wherein said chimeric natriuretic peptide is CD-NP.