WO2010051347A1 - A method for cardioprotection - Google Patents

Abstract

The present invention relates to the use of stanniocalcin-1 for cardioprotection, treating inflammation, reducing reactive oxidants, inducing expression of mitochondrial uncoupling proteins and promoting weight loss in a mammal.

Description

A METHOD FOR CARDIOPROTECTION

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claim priority to U.S. provisional patent application Serial No.

61/110,333 filed October 31, 2008, incorporated herein by reference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

[0002] The present invention was made in part with Government support under Grant Numbers P50 DK64233-01, DK062828 and DK62703-04 awarded by the National Institutes of Health, Bethesda, Maryland. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to the use of stanniocalcin-1 for cardioprotection, treating inflammation, reducing reactive oxidants, inducing expression of mitochondrial uncoupling proteins and promoting weight loss in a mammal.

[0004] The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography. [0005] Stanniocalcin-1 (STCl) is a 25 kDa homodimeric glycoprotein hormone involved in calcium regulation in bony fish (Fontaine, 1964), where elevation of serum calcium triggers the release of STCl from the corpuscles of Stannius (Stannius, 1939), organs associated with the kidneys (Wendelaar Bonga Se and Pang, P. K., (1991). On circulation in the gill and intestine, STCl inhibits calcium flux from the aquatic environment through these organs, thus maintaining normal calcium concentrations in the blood (Lafeber et al., 1988; Tagaki et al., 1985). In contrast to its restricted expression in bony fish, mammalian STCl is expressed in many tissues and organs (Chang et al., 1995; Varghese et al., 1988) and does not normally circulate in the blood (Wagner et al., 1997), and thus it is thought to function as an autocrine or paracrine substance. However, recent data suggest that mammalian STCl may be carried by red blood cells and appears to be filtered through the glomeruli (James et al., 2005). Moreover, unlike its well-defined role in serum calcium regulation in fish, little is known about its function in mammals. Current data suggest, however, that STCl may have a role in wound healing (Iyer et al., 1999), cellular metabolism (McCudden et al., 2002), atherogenesis (Sato et al., 1998), angiogenesis (Kahn et al., 2000), steroidogenesis (Paciga et al, 2003), muscle and bone development (Filvaroff et al., 2002; Yoshiko et al., 2002), phosphate uptake in kidney and gut (Madsen et al., 1998; Wagner et al., 1997), megakaryocyte differentiation (Serlachius et al., 2002), cancer biology (Chang et al., 2003), macrophage mobility and response to chemoattractants (Kanellis et al., 2004), energy production and superoxide production in macrophages (Wang et al., 2006) and trans-endothelial migration of macrophages and T cells (Chakraborty et al., 2007).

[0006] Increased oxidative stress is implicated in the pathology of a variety of diseases including metabolic, cardiovascular, neurological, liver, and pulmonary diseases. Oxidative stress is defined in general as excess formation and/or insufficient removal of highly reactive molecules such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Maritim et al 2003; Yorek, 2003). ROS include free radicals such as superoxide (*O₂ ^~), hydroxyl (*OH), peroxyl (*RO₂), hydroperoxyl (*HRθ₂ ^~) as well as nonradical species such as hydrogen peroxide (H₂O₂) and hydrochlorous acid (HOCl). ROS are continuously produced during normal physiologic processes, and are removed by the activity of antioxidant enzymes such as glutathione peroxidase, catalase, and superoxide dismutase. Under pathological conditions, ROS can be overproduced and result in oxidative stress. RNS include free radicals such as nitric oxide (*NO) and nitrogen dioxide (*N O₂ ^") as well as nonradicals such as peroxynitrite (ONOO^"), nitrous oxide (HNO₂), and alkyl peroxynitrates (RONOO). *NO₂ is normally produced from L-arginine by NO synthase (NOS). Three isoforms have been identified from three distinct genes: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). In the vascular endothelium, *NO mediates vasorelaxation by its acting on guanylate cyclase in vascular smooth muscle cells, initiating a cascade that leads to vasorelaxation. *NO also displays anti-proliferative properties and inhibits platelet and leukocyte adhesion to vascular endothelium. However, *NO easily reacts with superoxide (*O₂ ^"), generating the highly reactive molecule ONOO^" and triggering a cascade of harmful effects.

[0007] Chronic coronary artery disease is a leading cause of death in the Western world. Depending upon their duration and severity, single or intermittent ischemic episodes may lead to contractile dysfunction, myocardial cell injury and death. Although timely re-establishment of blood flow to an ischemic area is an obligatory requirement for myocardial salvage, there is evidence that reperfusion subsequent to ischemia triggers a series of cytotoxic and inflammatory events that can exacerbate irreversible tissue damage initiated during ischemia. Several lines of evidence suggest that a significant component of cellular injury associated with reperfusion is caused by a burst in the generation of highly reactive free radical oxygenase species (ROS), upon reoxygenation of the myocardium and subsequent activation of the inflammatory cascade (Cesselli et al, 2001; Bolli et al, 1995; Singh et al., 1995; Flaherty and Weisfeldt, 1988). The accumulation of ROS may eventually deplete the buffering capabilities of endogenous antioxidant systems, thereby exacerbating the deleterious effects of these reactive species. [0008] Despite advances in the treatment of ischemic heart disease, there remains a need for additional therapeutic methods.

SUMMARY OF THE INVENTION

[0009] The present invention relates to the use of stanniocalcin-1 for cardioprotection, treating inflammation, reducing reactive oxidants, inducing expression of mitochondrial uncoupling proteins and promoting weight loss in a mammal.

[0010] Thus, in one aspect, the present invention provides a method of protecting the heart in a mammal with hypertension or myocardial infarction. The method comprises administering a therapeutically effective amount of stanniocalcin-1, a stanniocalcin-1 derivative or a nucleic acid encoding stanniocalcin-1 or stanniocalcin-1 derivative to the mammal in need thereof to protect the heart.

[0011] In another aspect, the present invention provides a method of treating inflammation in a mammal in need thereof. The method comprises administering a therapeutically effective amount of stanniocalcin-1, a stanniocalcin-1 derivative or a nucleic acid encoding stanniocalcin-

1 or stanniocalcin-1 derivative to the mammal in need thereof to reduce inflammation. The mammal includes humans.

[0012] In an additional aspect, the present invention provides a method of reducing reactive oxidants in a mammal in need thereof. The method comprises administering a therapeutically effective amount of stanniocalcin-1, a stanniocalcin-1 derivative or a nucleic acid encoding stanniocalcin-1 or stanniocalcin-1 derivative to the mammal in need thereof to reduce reactive oxidants. The mammal includes humans.

[0013] In a further aspect, the present invention provides a method of inducing expression of mitochondrial uncoupling proteins in a mammal in need thereof. The method comprises administering a therapeutically effective amount of stanniocalcin-1, a stanniocalcin-1 derivative or a nucleic acid encoding stanniocalcin-1 or stanniocalcin-1 derivative to the mammal in need thereof to induce expression of mitochondrial uncoupling proteins. The mammal includes humans. [0014] In another aspect, the present invention provides a method of promoting weight in a mammal in need thereof. The method comprises administering a therapeutically effective amount of stanniocalcin-1, a stanniocalcin- 1 derivative or a nucleic acid encoding stanniocalcin- 1 or stanniocalcin-1 derivative to the mammal in need thereof to promote weight loss. The mammal includes humans.

BRIEF DESCRIPTION OF THE FIGURES

[0015] Figures IA and IB show inhibition of anti-GBM GN in STCl transgenic (STCl Tg) mice. Fig. IA: Periodic Acid Schiff s (PAS)- and Masson's trichrome-stained kidney tissue. Anti-GBM Ab injection to WT mice produced severe crescentic GN, characterized by extra- and intra-capillary mononuclear infiltration, hyaline cast formation (H), glomerulosclerosis and interstitial fibrosis (see trichrome staining). STCl Tg mice display less inflammation, crescent formation and fibrosis after anti-GBM. Arrow points to a crescent; G, glomeruli; H, hyaline casts in dilated tubules. Fig. IB: STCl Tg mice display fewer crescents, fewer glomerular endocapillary lesions, fewer sclerotic glomeruli, and less expansion in the tubulointerstitial compartment.

[0016] Figure 2 shows inhibition of crescentic GN in STCl Tg mice. WT mice had 3 -fold rise in BUN and 50% reduction in urine output; there were no significant changes in BUN or urine output in STCl Tg mice; baseline blood pressure was not different between WT and STCl Tg mice; however, it was significantly lower in STCl Tg mice after anti-GBM GN, consistent with renal protection.

[0017] Figures 3A-3D show effects in WT and STCl Tg mice. Fig. 3A shows Equal binding of sheep IgG to GBM in WT and STCl Tg mice. Ten days after anti-GBM Ab injection, frozen sections of kidney tissue were labeled with FITC-tagged anti-sheep IgG Ab. Equal binding of sheep anti-mouse GBM antibody to the GBM in both WT and STCl transgenic mice is illustrated (arrows point to glomeruli). Fig. 3B shows STCl levels in the serum. Western blot demonstrates higher level of STCl protein in the serum of STCl Tg mice compared to WT mice. Fig. 3 C shows STCl levels in the kidney. Western blot demonstrates equal expression of STCl in the kidneys (whole kidney lysates) of WT and STCl Tg mice after anti-GBM. (Fig. 3D shows STCl expression in macrophages. Dual staining for STCl (red) and the macrophage marker F4/80 (brown) demonstrates high level expression of STCl in kidney macrophages of STCl Tg mice compared with WT mice. [0018] Figure 4 shows expression of STCl in the endothelium. Immunohistochemistry studies reveal high level expression of STCl (brown) in the endothelium of STCl Tg mice (best appreciated in the glomerular capillaries), compared to WT mice. Also, expression of STCl in injured tubules of WT mice after anti-GBM GN. G, glomeruli; T, tubule.

[0019] Figure 5 shows that STCl Tg mice exhibit decreased infiltration of macrophages into the glomeruli following anti-GBM Ab injection. In a model of mouse anti-GBM GN, F4/80+ macrophages (resident) were absent from the glomeruli in both WT and STCl Tg mice; macrophage infiltration into the glomeruli was observed only in WT mice and the macrophages were predominantly F4/807CD68⁺ (inflammatory/"exudative"). T-cells (CD3) were predominantly interstitial and increased to a similar degree in WT and STCl Tg mice after anti- GBM GN.

[0020] Figures 6A-6D show that STCl transgenic mice exhibit decreased expression of MIP- 2, TGF-β and MCP-I, as well as MCP-I. Figs. 6A, 6B and 6C: Densities of respective bands corresponding to RNase Protection Assay were normalized to L32, and data represent the means & SE of at least 6 independent determinations. **, denotes P<0.01. Fig. 6D: Diminished expression of MCP-I protein in the kidneys of STCl Tg mice after anti-GBM GN, but no change in ICAM-I.

[0021] Figures 7A and 7B show effects on IgG levels. Fig. 7A: Decreased deposition of mouse C3 but not IgG in the glomeruli of STCl transgenic mice after anti-GBM GN. FITC- labeled anti-mouse C3 or IgG antibodies were used to determine the deposition of mouse C3 and IgG in the glomeruli of WT and STCl Tg mice 10 days after the administration of anti-GBM Ab. Upper panel shows representative images for C3; lower panels show representative images for mouse IgG. G, glomerulus. Fig. 7B: Serum IgG levels. Data represent the means + SEM of 5 independent determinations.

[0022] Figures 8A-8E show the effect of STCl on intracellular ATP. Fig. 8A: STCl decreases intracellular ATP levels. Raw264.7 cells grown in DMEM medium containing 10% FBS or freshly-isolated peritoneal macrophages suspended in DMEM plus 10% FBS, were treated with varying concentrations of STCl for 1 h. Values represent the mean + SEM of 3 independent experiments. Fig. 8B: Cells were treated with STCl (100 ng/ml) for increasing times. Fig. 8C: cells were treated with STCl (100 ng/ml) for 3h, washed in PBS (X3), and resuspended in fresh medium for 24 additional hours before ATP analysis (washout experiment). Fig. 8D: Cells were treated with denatured STCl (100 ng/ml) or vehicle for 3h. Data represent the mean + SEM of 3 independent experiments. Compared to vehicle: * denotes P<0.05; **, denotes PO.001; ***, denotes PO.0001. Fig. 8E: labeling for STCl is detected intracellularly in quiescent freshly isolated peritoneal macrophages. Ten min after the addition of recombinant STCl to the medium (100 ng/ml), intracellular labeling for STCl increases 10-fold, and colocalizes with the mitochondrial marker, mitotracker.

[0023] Figure 9 shows that STCl has no effect on the activity of complexes I-IV of the respiratory chain. Raw264.7 cells grown in DMEM medium containing 10% FBS were treated with STCl (100 ng/ml) for 3 h, lysed and analyzed for respiratory chain complex activity as described in methods. Data represent the mean + SEM of three independent experiments. Differences were not statistically significant.

[0024] Figures 1OA and 1OB show that STCl induces UCP2 expression in macrophages. Fig. Raw264.7 cells grown in DMEM medium containing 10% FBS were treated with STCl (100 ng/ml) for varying periods of time. Cells were lysed in modified RIPA buffer and UCP2 protein abundance was determined using Western blotting. Fig. 1OA: Representative blot is shown. Fig. 1OB: Graph represents the mean + SEM of 3 independent experiments. Densitometry values of UCP2 were normalized to GAPDH. *, PO.05 compared to Oh; **, PO.01 compared to Oh. [0025] Figure 11 shows that STCl decreases mitochondrial membrane potential. Raw264.7 cells grown in DMEM medium containing 10% FBS were treated with STCl (150 ng/ml) for 5 and 24 h followed by mitochondrial membrane potential measurement using JC-I florescence- based assay (Red/Green fluorescence ratio). Data represent the mean + SEM of 8 independent experiments. * denotes P<0.001.

[0026] Figure 12 shows that STCl -induced reduction in superoxide is UCP2-dependent. Freshly-isolated peritoneal macrophages from WT and UCP2^7" mice cells were suspended in DMEM medium containing 10% FBS. Sixteen hours prior to the experiment, cells were shifted to 1% FBS-containing medium, then were treated with STCl (100 ng/ml) for 24 additional hours. Fifteen minutes prior to completion of the experiment, cells were treated with DHE and red fluorescence (ethidium bromide) was quantitated using flow cytometry. Bar graph represents the mean + SEM of 3 independent experiments. * denotes P<0.05.

[0027] Figures 13A and 13 B show that STCl enhances cell viability. Fig. 13A: Raw264.7 cells grown in DMEM medium containing 10% FBS, were shifted to DMEM containing 1% FBS for sixteen hours, followed by treatment with STCl (100 ng/ml) in DMEM containing 1% FBS for 24 additional hours. Medium was then collected for LDH analysis. Data represent the mean + SEM of three independent experiments. * denotes P<0.01. Fig. 13B: Raw264.7 cells were grown in DMEM medium containing 10% FBS. Cells were then shifted to DMEM containing 1% FBS in the presence of STCl (100 ng/ml) or vehicle for 48 h. Cells were harvested using cell lifter, fixed in 70% ethanol, stained with propidium iodide and treated with

RNase for 30 minutes. Cell cycle analysis was performed using flow cytometry. Peak F represents apoptotic cells; peak C represents cells in Gl phase; peak D represents cells in the S- phase; peak E represents cells in G2/M phase. The differences between peaks F and C were statistically significant when comparing STCl -treated cells vs controls. * denotes P<0.0001.

[0028] Figure 14 shows that ATP levels are decreased in heart lysates of STCl Tg mice.

[0029] Figure 15 shows that transgenic overexpression of STCl does not affect the activities of respiratory chain complexes in the heart (N=3, p=NS).

[0030] Figure 16 shows that STCl increases the expression of UCP3 protein in cultured adult rat cardiomyocytes.

[0031] Figure 17 shows that STCl diminishes angiotensin-II-mediated increase in superoxide generation in cultured adult rat cardiomyocytes. Red depicts DHE staining in cardiomyocytes, which corresponds to superoxide generation.

[0032] Figure 18 shows increased STCl protein expression in the heart after aortic banding

(n=7).

[0033] Figure 19 shows increased UCP3 protein expression in the heart after aortic banding.

[0034] Figure 20 shows increased STCl expression in blood vessels and cardiomyocytes after aortic banding: gradual increase toward the periphery. Similar distribution was observed with UCP3.

[0035] Figure 21 shows increased STCl expression in blood vessels and mononuclear cells after ischemia/reperfusion, suggesting involvement in the repair process following ischemic injury. Similar distribution were observed with UCP3.

[0036] Figure 22 shows effects of STCl on UCP3 expression and Angiotensin-II medicated increase in superoxide generation. Left panel: STCl increases the expression of UCP3 protein in cultured adult rat cardiomyocytes. Right panel: STCl diminishes A-II-mediated increase in superoxide generation in cultured adult rat cardiomyocytes. Red, depicts DHE staining.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention relates to the use of stanniocalcin-1 for cardioprotection, treating inflammation, reducing reactive oxidants, inducing expression of mitochondrial uncoupling proteins and promoting weight loss in a mammal. [0038] Treating" or "treatment" of any disease or disorder refers to arresting or ameliorating a disease, disorder, or at least one of the clinical symptoms of a disease or disorder, reducing the risk of acquiring a disease, disorder, or at least one of the clinical symptoms of a disease or disorder, reducing the development of a disease, disorder or at least one of the clinical symptoms of the disease or disorder, or reducing the risk of developing a disease or disorder or at least one of the clinical symptoms of a disease or disorder. "Treating" or "treatment" also refers to inhibiting the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both, and to inhibiting at least one physical parameter which may or may not be discernible to the patient. In certain embodiments, "treating" or "treatment" refers to delaying the onset of the disease or disorder or at least one or more symptoms thereof in a patient which may be exposed to or predisposed to a disease or disorder even though that patient does not yet experience or display symptoms of the disease or disorder.

[0039] The present invention contemplates the use of the protein stanniocalcin-1, a derivative of this protein or a nucleic acid encoding stanniocalcin- 1. In one embodiment, stanniocalcin- 1 has the protein sequence set forth in GenBank Accession No. NP_003146. In another embodiment, stanniocalcin-1 has the protein sequence set forth as amino acids 18-247 of GenBank Accession No. NP_003146. In one embodiment, stanniocalcin-1 is encoded by a nucleic acid having the sequence set forth in GenBank Accession No. NM_003155. In some embodiments, a derivative of stanniocalcin- 1 includes conventional protein derivatives, such as those described herein. In another embodiment, stanniocalcin-1 is encoded by a nucleic acid having the sequence set forth as nucleotides 285-1028 of GenBank Accession No. NM_003155. In an additional embodiment, stanniocalcin-1 is encoded by a nucleic acid having the sequence set forth as nucleotides 285-1025 of GenBank Accession No. NM_003155. In a further embodiment, stanniocalcin-1 is encoded by a nucleic acid having the sequence set forth as nucleotides 336-1028 of GenBank Accession No. NM_003155. In an additional embodiment, stanniocalcin-1 is encoded by a nucleic acid having the sequence set forth as nucleotides 336- 1025 of GenBank Accession No. NM_003155.

[0040] Thus, in one aspect, the present invention provides a method of protecting the heart (cardioprotection) in a mammal with hypertension or myocardial infarction. The method comprises administering a therapeutically effective amount of stanniocalcin-1, a derivative thereof or a nucleic acid encoding stanniocalcin-1 or stanniocalcin-1 derivative to the mammal in need thereof to protect the heart. In one embodiment, cardioprotection is provided by reducing inflammation and superoxide generation in a mammal having hypertension. In another embodiment, cardioprotection is provided by reducing inflammation and superoxide generation in a mammalian suffering a myocardial infarction. In an additional embodiment, the cardioprotection is provided by suppressing angiotensinogen-II-mediated superoxide generation in a mammal having inflammatory artery disease. In these embodiments, a therapeutically effective amount of stanniocalcin- 1 is an amount of stanniocalcin- 1 or stanniocalcin- 1 derivative that is administered to the mammal as a protein or produced by in vivo expression of a nucleic acid that is administered to a mammal to reduce inflammation and superoxide generation in the heart tissue. In one embodiment, the heart tissue is contacted with stannioclacin-1, a stanniocalcin- 1 derivative or with a nucleic acid encoding stanniocalcin- 1 or stanniocalcin- 1 derivative. In another embodiment, stanniocalcin- 1, a stanniocalcin- 1 derivative or with a nucleic acid encoding stanniocalcin- 1 or stanniocalcin- 1 derivative can be administered topically, transdermally, subcutaneously, intra-nasally, intraveneously, parenterally, systemically or by tablet.

[0041] In another aspect, the present invention provides a method of treating inflammation in a mammal in need thereof. The method comprises administering a therapeutically effective amount of stanniocalcin- 1, a stanniocalcin- 1 derivative or a nucleic acid encoding stanniocalcin- 1 or stanniocalcin- 1 derivative to the mammal in need thereof to reduce inflammation. In one embodiment, the inflamed tissue or inflamed area of the mammal is contacted with stannioclacin-1, a stanniocalcin- 1 derivative or a nucleic acid encoding stanniocalcin- 1 or stanniocalcin- 1 derivative. In another embodiment, stanniocalcin- 1, a stanniocalcin- 1 derivative or a nucleic acid encoding stanniocalcin- 1 or stanniocalcin- 1 derivative can be administered topically, transdermally, subcutaneously, intra-nasally, intraveneously, parenterally, systemically or by tablet.

[0042] Inflammation in the subject may be associated with any one or more of various conditions or diseases. For example, the inflammation in the subject may be due to a wound, to periodontal disease, to delayed-type hypersensitivity, to autoimmune disease, or to arthritis. The subject may be suffering from an autoimmune disease. In one embodiment, the subject is suffering from multiple sclerosis, autoimmune encephalitis, lupus nephritis, or autoimmune complications from diabetes. The subject may be suffering from diabetes, for example, type I diabetes. The subject may be suffering from Behchet's syndrome. The subject may be suffering from Sjogren's syndrome. The subject may be suffering from colitis, ulcerative colitis, inflammatory colitis, Crohn's disease or the like. The subject may be suffering from arthritis which is osteoarthritis, rheumatoid arthritis, collagen-induced arthritis, psoriatic arthritis, lupus- induced arthritis, or trauma-induced arthritis. The subject may be suffering from another overall condition or disease which includes a manifestation of inflammation, such as arthritis. In another embodiment, the subject is suffering from an allergy or is experiencing an allergic response. In one embodiment, the subject is suffering from asthma. The subject may be suffering from allergic asthma. In another embodiment, the subject is suffering from systemic lupus erythematosus, inflammatory lupus nephritis, septic shock or endotoxemia. [0043] In an additional aspect, the present invention provides a method of reducing reactive oxidants in a mammal in need thereof. The method comprises administering a therapeutically effective amount of stanniocalcin-1, a stanniocalcin- 1 derivative or a nucleic acid encoding stanniocalcin- 1 or stanniocalcin- 1 derivative to the mammal in need thereof to reduce reactive oxidants. In one embodiment, stanniocalcin-1, a stanniocalcin-1 derivative or with a nucleic acid encoding stanniocalcin-1 or stanniocalcin-1 derivative can be administered topically, transdermally, subcutaneous Iy, intra-nasally, intraveneously, parenterally, systemically or by tablet.

[0044] In a further aspect, the present invention provides a method of inducing expression of mitochondrial uncoupling proteins in a mammal in need thereof. The method comprises administering a therapeutically effective amount of stanniocalcin-1, a stanniocalcin-1 derivative or a nucleic acid encoding stanniocalcin-1 or stanniocalcin-1 derivative to the mammal in need thereof to induce expression of mitochondrial uncoupling proteins. In some embodiments, the induction of expression of mitochondrial uncoupling proteins is in cells of the mammals. In one embodiment, the cells are macrophages. In another embodiment, the cells are heart cells and macrophages. In one embodiment, stanniocalcin-1, a stanniocalcin-1 derivative or with a nucleic acid encoding stanniocalcin-1 or stanniocalcin-1 derivative can be administered topically, transdermally, subcutaneously, intra-nasally, intraveneously, parenterally, systemically or by tablet.

[0045] In another aspect, the present invention provides a method of promoting weight in a mammal in need thereof. The method comprises administering a therapeutically effective amount of stanniocalcin-1, a stanniocalcin-1 derivative or a nucleic acid encoding stanniocalcin- 1 or stanniocalcin-1 derivative to the mammal in need thereof to promote weight loss. In one embodiment, the stanniocalcin- 1 , a derivative thereof or a nucleic acid encoding stanniocalcin- 1 is administered topically, subcutaneously, intra-nasally or by tablet. In one embodiment, the therapeutically effective amount of stanniocalcin-1, a derivative thereof or a nucleic acid encoding stanniocalcin- 1 or stanniocalcin- 1 derivative is an amount that decreases the efficiency of food utilization as a result of the uncoupling of the mitochondria and less efficiency in energy utilization.

[0046] Proteins and fusion proteins of this invention may be produced by any technique known per se in the art, such as by recombinant technologies, chemical synthesis, cloning, ligations, or combinations thereof. In a particular embodiment, the proteins or fusion proteins are produced by recombinant technologies, e.g., by expression of a corresponding nucleic acid in a suitable host cell and recovering the polypeptide produced. The protein produced may be glycosylated or not, or may contain other post-translational modifications depending on the host cell type used.

[0047] The vectors to be used in the method of producing a polypeptide according to the present invention can be episomal or non-/homologously integrating vectors, which can be introduced into the appropriate host cells by any suitable means (transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc.). Factors of importance in selecting a particular plasmid, viral or retroviral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species. The vectors should allow the expression of the polypeptide or fusion proteins of the invention in prokaryotic or eukaryotic host cells, under the control of appropriate transcriptional initiation/termination regulatory sequences, which are chosen to be constitutively active or inducible in said cell. A cell line substantially enriched in such cells can be then isolated to provide a stable cell line.

[0048] Host cells are transfected or transformed with expression or cloning vectors described herein for protein production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Sambrook and Russell, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001); Ausubel et ah, Current Protocols in Molecular Biology (John Wiley & Sons, updated through 2008). [0049] For eukaryotic host cells (e.g. yeasts, insect or mammalian cells), different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived form viral sources, such as adenovirus, papilloma virus, Simian virus or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Examples are the TK promoter of the Herpes virus, the SV40 early promoter, the yeast gal4 gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for repression and activation, so that expression of the genes can be modulated. The cells which have been stably transformed by the introduced DNA can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may also provide for phototrophy to an auxotrophic host, biocide resistance, e.g. antibiotics, or heavy metals such as copper, or the like. The selectable marker gene can be either directly linked to the DNA sequences to be expressed (e.g., on the same vector), or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins of the invention.

[0050] Particularly suitable prokaryotic cells include bacteria (such as Bacillus subtilis or Escherichia colϊ) transformed with a recombinant bacteriophage, plasmid or cosmid DNA expression vector. Preferred cells to be used for producing the proteins or fusion proteins of he present invention are eukaryotic host cells, e.g. mammalian cells, such as human, monkey (e.g. COS cells), mouse, and Chinese Hamster Ovary (CHO) cells, because they provide post- translational modifications to protein molecules, including correct folding or glycosylation at correct sites. Alternative eukaryotic host cells are yeast cells (e.g., Saccharomyces , Kluyveromyces , etc.) transformed with yeast expression vectors. Also yeast cells can carry out post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids that can be utilized for production of the desired proteins in yeast. Yeast cells recognize leader sequences in cloned mammalian gene products and secrete polypeptides bearing leader sequences (i.e., pre-peptides).

[0051] For long-term, high-yield production of a recombinant polypeptide, stable expression is preferred. For example, cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1 -2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. A cell line substantially enriched in such cells can be then isolated to provide a stable cell line. [0052] Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney (BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example Hep G2) cells and a number of other cell lines. In the baculovirus system, the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen.

[0053] In addition to recombinant DNA technologies, the polypeptides or fusion proteins of this invention may be prepared by chemical synthesis technologies. Examples of chemical synthesis technologies are solid phase synthesis and liquid phase synthesis. As a solid phase synthesis, for example, the amino acid corresponding to the carboxy -terminus of the polypeptide to be synthesised is bound to a support which is insoluble in organic solvents and, by alternate repetition of reactions (e.g., by sequential condensation of amino acids with their amino groups and side chain functional groups protected with appropriate protective groups), the polypeptide chain is extended. Solid phase synthesis methods are largely classified by the tboc method and the Fmoc method, depending on the type of protective group used.

[0054] The protein of the present invention can be produced, formulated, administered, or generically used in other alternative forms that can be preferred according to the desired method of use and/or production. The proteins of the invention can be post-translationally modified, for example by glycosylation. The polypeptides or proteins of the invention can be provided in isolated (or purified) biologically active form, or as precursors, derivatives and/or salts thereof. Said biological activity is at least one of the biological activities described herein. [0055] Muteins, analogs or active fragments, of the foregoing protein are also contemplated here. See, e.g., Hammerland et al. (1992). Derivative muteins, analogs or active fragments of the foregoing protein may be synthesized according to known techniques, including conservative amino acid substitutions, such as outlined in U.S. Patents No. 5,545,723 (see particularly col. 2, line 50 to col. 3, line 8); 5,534,615 (see particularly col. 19, line 45 to col. 22, line 33); and 5,364,769 (see particularly col. 4, line 55 to col. 7, line 26), each incorporated herein by reference. Pegylated derivatives are also contemplated and are prepared according to known techniques, such as described by Kodera et al. (1998), Roberts et al. (2002) and Veronese and Harris (2002). Proteins with modified glycosylation patterns can also be produced using conventional techniques, e.g., by modifying the coding sequence for stanniocalcin-1 to eliminate glycosylation sites or to insert glycosylation sites.

[0056] Precursors" are compounds which can be converted into the polypeptides of present invention by metabolic and/or enzymatic processing prior to or after administration thereof to cells or an organism. The term "salts" herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the proteins of the present invention. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid. Thus, examples of acid addition salts include, but are not limited to, hydrochloride, sulphates, nitrates or phosphates and acetates, trifluoroacetates, propionates, succinates, benzoates, citrates, tartrates, fumarates, maleates, methane-sulfonates, isothionates, theophylline acetates, salicylates, or the like. Any of such salts should have substantially similar activity to the proteins of the invention. The term "derivatives" as used herein refers to derivatives that can be prepared from the functional groups present on the lateral chains of the amino acid moieties or on the amino-/or carboxy-terminal groups according to methods known per se in the art. Such derivatives include for example esters or aliphatic amides of the carboxyl-groups and N-acyl derivatives of free amino groups or O-acyl derivatives of free hydroxyl-groups and are formed with acyl-groups as for example alcanoyl- or aroyl- groups. Purification of the polypeptides of the invention can be carried out by a variety of methods known per se in the art, such as, without limitation, any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like. A particular purification procedure is affinity chromatography, using (monoclonal) antibodies or affinity groups which selectively bind the polypeptide and which are typically immobilized on a gel matrix contained within a column. Purified preparations of the proteins of the invention, as used herein, refers to preparations which contain less than 15% of contaminants, more preferably which comprise at least 90, 95 or 97% of the polypeptide.

[0057] Useful conjugates or complexes can also be generated for improving the agents in terms of drug delivery efficacy. For this purpose, the protein or fusion proteins of the invention can be in the form of active conjugates or complex with molecules such as polyethylene glycol and other natural or synthetic polymers (Harris and Chess, 2003; Greenwald et al., 2003; Pillai and Panchagnula, 2001). In this regard, the present invention contemplates chemically modified proteins as disclosed herein, in which the protein is linked with a polymer. Typically, the polymer is water soluble so that the conjugate does not precipitate in an aqueous environment, such as a physiological environment. An example of a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation. In this way, the degree of polymerization can be controlled. An example of a reactive aldehyde is polyethylene glycol propionaldehyde, ormono-(Cl-ClO) alkoxy, or aryloxy derivatives thereof (see, for example, U.S. Patent No. 5,252,714). The polymer may be branched or unbranched. Moreover, a mixture of polymers can be used to produce the conjugates. The conjugates used for therapy can comprise pharmaceutically acceptable water- soluble polymer moieties. Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono-(Cl-ClO) alkoxy -PEG, aryloxy -PEG, poly-(N-vinyl pyrrolidone) PEG, tresyl monomethoxy PEG, PEG propionaldehyde, bis-succinimidyl carbonate PEG, propylene glycol homopolymers, a polypropyleneoxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers. Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000. A conjugate can also comprise a mixture of such water-soluble polymers.

[0058] Pharmaceutical compositions containing a protein of the present invention as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, 2005.

[0059] The pharmaceutical compositions of the present invention may contain, in combination with the proteins of the invention as active ingredient, suitable pharmaceutically acceptable diluents, carriers, biologically compatible vehicles and additives which are suitable for administration to an animal (for example, physiological saline solution) and optionally comprising auxiliaries (like excipients, stabilizers, or adjuvants) which facilitate the processing of the active compounds into preparations which can be used pharmaceutically. As used herein, the term "pharmaceutically acceptable" carrier means a non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations.

[0060] The pharmaceutical compositions may be formulated in any acceptable way to meet the needs of the mode of administration. For example, the use of biomaterials and other polymers for drug delivery, as well the different techniques and models to validate a specific mode of administration, are disclosed in literature (Luo and Prestwich, 2001; Cleland et al, 2001). "Pharmaceutically acceptable" is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which is administered. For example, for parenteral administration, the above active ingredients may be formulated in unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution. Carriers can be selected also from starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the various oils, including those of petroleum, animal, vegetable or synthetic origin (peanut oil, soybean oil, mineral oil, sesame oil). [0061] The pharmaceutical composition may be in a liquid or lyophilized form and comprises a diluent (Tris, citrate, acetate or phosphate buffers) having various pH values and ionic strengths, solubilizer such as Tween or Polysorbate, carriers such as human serum albumin or gelatin, preservatives such as thimerosal, parabens, benzylalconium chloride or benzyl alcohol, antioxidants such as ascrobic acid or sodium metabisulfite, and other components such as lysine or glycine. Selection of a particular composition will depend upon a number of factors, including the condition being treated, the route of administration and the pharmacokinetic parameters desired. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. Examples of pharmaceutically acceptable antioxidants include, but are not limited to, water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, and the like; oil soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and the metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like. A more extensive survey of components suitable for pharmaceutical compositions is found in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, 2005.

[0062] A variety of administration routes are available. The particular mode selected will depend of course, upon the particular drug selected, the severity of the disease state being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, sublingual, topical, nasal, transdermal or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, epidural, irrigation, intramuscular, release pumps, or infusion. For example, administration of the active agent according to this invention may be achieved using any suitable delivery means, including those described in U.S. Patent No. 5,844,077, incorporated herein by reference. Subcutaneous administration can be accomplished using a pharmaceutical composition comprising the effective amount of the active agent in a pharmaceutically acceptable depot that releases the active agent in the desired dosage per day. [0063] Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.

[0064] The active agent, which includes the foregoing protein, can also be administered in a cell based delivery system in which a DNA sequence encoding an active agent is introduced into cells designed for implantation in the body of the patient. Suitable delivery systems are described in U.S. Patent No. 5,550,050 and published PCT Application Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635. Suitable DNA sequences can be prepared synthetically for each active agent on the basis of the developed sequences and the known genetic code. [0065] The pharmaceutical compositions of the present invention can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, and the like, for the prolonged administration of the protein at a predetermined rate, preferably in unit dosage forms suitable for single administration of precise dosages.

[0066] Parenteral administration can be by bolus injection or by gradual perfusion over time. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients known in the art, and can be prepared according to routine methods. In addition, suspension of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions that may contain substances increasing the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.

[0067] The active agent is preferably administered in a therapeutically effective amount. By a "therapeutically effective amount" or simply "effective amount" of an active compound is meant a sufficient amount of the compound to treat the desired condition at a reasonable benefit/risk ratio applicable to any medical treatment. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington: The Science and Practice of Pharmacy.

[0068] It is understood that the dosage administered will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art. The total dose required for each treatment may be administered by multiple doses or in a single dose. Thus, "Pharmaceutical composition" means physically discrete coherent portions suitable for medical administration. "Pharmaceutical composition in dosage unit form" means physically discrete coherent units suitable for medical administration, each containing a daily dose or a multiple (up to four times) or a sub-multiple (down to a fortieth) of a daily dose of the active compound in association with a carrier and/or enclosed within an envelope. Whether the composition contains a daily dose, or for example, a half, a third or a quarter of a daily dose, will depend on whether the pharmaceutical composition is to be administered once or, for example, twice, three times or four times a day, respectively.

[0069] Dosage may be adjusted appropriately to achieve desired drug levels, locally or systemically. Typically the active agents of the present invention exhibit their effect at a dosage range from about 10 ng/ml (10 ng/g) to about 500 ng/ml (500 ng/g), preferably from about 50 ng/ml (50 ng/g) to about 200 ng/ml (200 ng/g) of the active ingredient, more preferably from about 50 ng/ml (50 ng/g) to about 100 ng/ml (100 ng/g). A suitable dose can be administered in multiple sub-doses per day. Dosages are generally initiated at lower levels and increased until desired effects are achieved. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Continuous dosing over, for example, 24 hours or multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.

[0070] Advantageously, the compositions are formulated as dosage units, each unit being adapted to supply a fixed dose of active ingredients. Tablets, coated tablets, capsules, ampoules and suppositories are examples of dosage forms according to the invention. [0071] It is only necessary that the active ingredient constitute an effective amount, i.e., such that a suitable effective dosage will be consistent with the dosage form employed in single or multiple unit doses. The exact individual dosages, as well as daily dosages, are determined according to standard medical principles under the direction of a physician or veterinarian for use humans or animals.

[0072] The pharmaceutical compositions will generally contain from about 0.0001 to 99 wt. %, preferably about 0.001 to 50 wt. %, more preferably about 0.01 to 10 wt.% of the active ingredient by weight of the total composition. In addition to the active agent, the pharmaceutical compositions and medicaments can also contain other pharmaceutically active compounds. Examples of other pharmaceutically active compounds include, but are not limited to, analgesic agents, cytokines and therapeutic agents in all of the major areas of clinical medicine. When used with other pharmaceutically active compounds, the protein of the present invention may be delivered in the form of drug cocktails. A cocktail is a mixture of any one of the compounds useful with this invention with another drug or agent. In this embodiment, a common administration vehicle (e.g., pill, tablet, implant, pump, injectable solution, etc.) would contain both the instant composition in combination with a supplementary potentiating agent. The individual drugs of the cocktail are each administered in therapeutically effective amounts. A therapeutically effective amount will be determined by the parameters described above; but, in any event, is that amount which establishes a level of the drugs in the area of body where the drugs are required for a period of time which is effective in attaining the desired effects. [0073] The proteins of the present invention may be employed in accordance with the present invention by expression of such proteins in vivo, which is often referred to as "gene therapy." Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a protein of the present invention. Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle. [0074] Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

[0075] The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter, or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and -actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B 19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

[0076] The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the gene encoding the protein.

[0077] The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, -2, -AM, PA12, T19-14X, VT-19-17-H2, CRE, CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller (1990). The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO₄ precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

[0078] The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

[0079] Alternatively, the gene transfer method described in U.S. Patent Application Publication No. 2008/0261909 can be used for in vivo delivery to organs. This method can be used for hearts as well as in other thin-walled organs or structures, such as hollow organs, including gastrointestinal organs or reproductive organs. The organ can be, for example, the stomach, gall bladder, small intestine, large intestine, rectum, uterus, or urinary bladder. The organ may be any thin-walled organ, including but not limited to the eye, skin, diaphragm, and lung. The method can be used, inter alia, for humans undergoing cardiac surgical procedures. Thoracoscopic delivery can optionally be used to substantially reduce the invasive nature and increase subject tolerance.

[0080] A further alternative for in vivo gene delivery involves the use of gene-loaded microbubbles, no larger than red blood cells, that can be injected into the patient's bloodstream, tracked via ultrasound imaging, and then ruptured by a focused ultrasound pulse to release their gene payload when they reach the desired spot. Because the genes are only released at the site of the diseased tissue, the patient's total body exposure to them could be limited. This technique has been used to deliver gene constructs to various tissues. See, for example, Lawrie et al. (2000), Manome et al. (2000) and Anwer et al. (2000).

[0081] The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1982); Sambrook et al, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989); Sambrook and Russell, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001); Ausubel et al, Current Protocols in Molecular Biology (John Wiley & Sons, updated through 2008); Glover, 1985, DNA Cloning (IRL Press, Oxford); Russell, 1984, Molecular biology of plants: a laboratory course manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Fire et al, RNA Interference Technology: From Basic Science to Drug Development, Cambridge University Press, Cambridge, 2005; Schepers, RNA Interference in Practice, Wiley- VCH, 2005; Engelke, RNA Interference (RNAi): The Nuts & Bolts of siRNA Technology, DNA Press, 2003; Gott, RNA Interference, Editing, and Modification: Methods and Protocols (Methods in Molecular Biology), Human Press, Totowa, NJ, 2004; Sohail, Gene Silencing by RNA Interference: Technology and Application, CRC, 2004.

EXAMPLES

[0082] The present invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.

EXAMPLE 1

Experimental Procedures for Examples 2-6

[0083] Materials: Sheep anti-mouse GBM antibody was a gift from Dr Hui Lan (University of Hong Kong). Polyclonal rabbit anti-STCl antibodies were a gift from Dr Gert FHk (Wendelaar Bonga et al., 1989). Antibodies for mouse pan-macrophage marker (CD68) were purchased from AbD Serotec (Raleigh, NC). Actin antibodies were purchased from Chemicon (Temecula, CA). ICAM-I antibodies were purchased from Santa Cruz. MCP-I antibodies were previously described (Li et al., 2005). Antibodies for mouse macrophage marker F4/80 and T- lymphocytes (CD3) were purchased from BD PharMingen (San Diego, CA). FITC-tagged monoclonal antibodies for sheep IgG were purchased from R&D systems (Minneapolis, MN). FITC-tagged antibodies for mouse C3 were purchased from Cedarlane Laboratories (Ontario, Canada). FITC-tagged antibodies for mouse IgG were purchased from Sigma (St. Louis, MO). ECL plus reagent and horseradish peroxidase-linked anti-mouse IgG were purchased from Amersham (Little Chalfont, Buckinghamshire, UK). All other chemicals and reagents were purchased from Sigma. Serum levels of IgG were determined using mouse IgG ELISA Kit as per manufacturer instructions (BETHYL Bethyl Laboratories, Montgomery, TX). [0084] Anti-GBM glomerulonephritis model: Transgenic overexpression of STCl is driven by the mouse metallothionein I (MT-I) minimal promoter over C57B/6CBA genetic background, and displays strong preferential expression of STCl transgene mRNA in the liver, heart and brain, and low in numerous other tissues including the kidneys (Kanellis et al., 2004); however, our data show STCl is preferentially expressed in macrophages and endothelial cells, where it is best appreciated in the glomerular capillaries. STCl is detectable in the serum of wild type mice, and as expected serum levels of STCl in transgenic mice were significantly higher (see data and Kanellis et al., 2004). Twenty five gram, one year old male mice were used; they were placed on ad-lib food and water intake throughout the experiment. Accelerated anti-GBM GN was induced in transgenic and wild type mice by subcutaneous "priming" with 1 mg/mouse of normal sheep IgG in Freund's complete adjuvant (FCA), followed 7 days later by an IV injection of 0.2 mg/g of sheep anti-mouse GBM antibody. Mice were euthanized 10 days after anti-GBM antibody injection.

[0085] Blood pressure measurement and renal function assessment: Systolic blood pressure was recorded by tail plethysmography using the BP2000 blood pressure analysis system (Visitech Systems, Inc., Apex, NC) in conscious mice at baseline and prior to euthanasia. Urinary protein concentrations were determined by the Bradford method, adapted to a microtiter plate assay. Coomassie reagent (USB, Cleveland, OH) was added to the diluted urine samples. After 10 minutes, the absorbance at 595-nm wavelength was read on ELX800 microplate reader (Bio-Tek Instruments, VT). The protein concentrations were calculated by reference to bovine serum albumin standards (Sigma).

[0086] Serum urea measurement: serum samples were treated with urease (US Biochemical Corp., Cleveland, OH) and the resultant ammonia was reacted with O-phthaldialdehyde/2- mercaptoethanol reagent (Sigma; St Louis, MO) in phosphate buffer (pH 7.4) for 30 min. Urea was measured as fluorescence (excitation at 405 nm and emission at 455 nm). [0087] Morphometric analysis: Tissue sections were evaluated by a kidney pathologist who was unfamiliar with the experimental protocol. Interstitial volume was determined using a point- counting technique on trichrome-stained sections. The interstitial volume was expressed as the percentage of grid points of a 1-cm² graded ocular grid viewed at X20 magnification, which lay within the interstitial area. Five to ten random fields were used for morphometry. Crescentic formation was counted from more than 100 glomeruli for each mouse and expressed as the percentage of positive glomeruli out of the total number examined. Total macrophages (CD68 ), resident macrophages (F4/80 ) and T-cells (CD3 ) infiltrating the glomeruli and interstitium were counted, and the results were expressed as total cell number per glomerulus, or 10 interstitial grids (1-cm graded ocular grids viewed at X20 magnification), respectively. The extent of glomerular sclerosis was expressed as % of Periodic Acid Schiff (PAS)-staining positive area per whole glomerular area. Each area was measured by tracking the glomerular tuft aided by computer manipulation using Mac Scope version 6.02 (Mitani Shoji Co., Ltd., Fukui, Japan). The extent of interstitial fibrosis was determined by Masson's trichrome, and is based on survey of the whole area of the cortex in the individual kidney sections and expressed as a percentage of the field using Mac Scope version 6.02.

[0088] Immunohistochemistry: Ten days after injection of anti-GBM antibody, kidney tissue was fixed in 10% formaldehyde followed by dehydration in graded alcohols and embedding in paraffin blocks using standard techniques. Five μm sections were cut, dried and rehydrated. Labeling was carried out using polyclonal rabbit anti-STCl (1: 1000 dilution; Wendelaar Bonga et al, 1989) or monoclonal rat anti-mouse F4/80 antigen antibodies (1 : 100 dilution), and detection was carried out using peroxidase enzyme-based detection system (Vector Laboratories). Control for labeling was carried out in the presence of non-immune IgG, and showed no staining. Staining with anti-CD68 (1: 100 dilution), anti-CD3 (1 :50 dilution), FITC- tagged anti-sheep IgG (1:80 dilution), and FITC-tagged anti-mouse IgG (1 :50 dilution) was carried out using frozen sections (5 μm thickness), applying standard techniques. Photomicrographs were taken using Labophot-2 Nikon microscope with a MagnaFire Olympus digital camera.

[0089] Western blotting: Kidney tissue was homogenized using (Polytron, for 30 seconds) in a modified RIPA buffer [150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, containing 1 mM phenylmethylsulfonylfluoride and 1 μg/ml leupeptin] and centrifuged for 10 min at 1400 rpm/4° C to remove cell debris. Fifty μg of total kidney lysates or sera were resolved on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and incubated with antibodies against: actin; STCl (1: 1000 dilution); ICAM-I (1: 1000 dilution) or MCP-I (1: 1000 dilution). After washing with TBST buffer (20 mM Tris, pH 7.6, 137 mM NaCl, 0.1% Tween-20), the membrane was incubated with horseradish peroxidase-conjugated anti-goat IgG. The bound antibodies were visualized using chemiluminescence.

[0090] RN Ase protection assay: Riboprobes for GATA-3, IL-6, IL-10, IL- 12, IL- 18, IFN-γ, MCP-I, MIF, MIP -2, RANTES, TGF-β, TNF-α, TCA-3, GAPDH and the ribosomal protein L- 32 were generated by PCR reaction using cDNA templates. Total RNA was isolated from whole kidneys of WT and STCl Tg mice ten days after anti-GBM Ab injection, using RNAzol (Tel- Test, Friendswood, TX). Three micrograms of total RNA from each sample were used in RNase protection assay using the Torrey Pines Biolabs kit (Houston, TX, USA) as previously described 24. Phosphoimage quantitation was performed using the Phosphorlmager SI scanning instrument and ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA, USA). [0091] Statistical analysis: Data were expressed as mean ±SEM. Statistical significance was determined by unpaired t-test. A P value less than 0.05 was considered statistically significant. [0092] Nonstandard abbreviations used: STCl, stanniocalcin- 1 ; GBM, glomerular basement membrane; BUN, blood urea nitrogen; PAS, Periodic Acid Schiff; C3, complement component 3; FCA, Freund's complete adjuvant; CD3, cluster of differentiation-3, a marker of T-cells; MCP-I Macrophage chemotactic protein-1; CCL2, Chemokine (C-C motif) ligand 2; MIF, macrophage migration inhibitory factor; MIP, macrophage inflammatory protein; CX3CR1, receptor specific for the chemokine, fractalkine; GR-I, a granulocyte marker known also as Ly- 6G; CCR2, C-C motif chemokine receptor-2; CD62L, L-selectin; MP20, membrane protein 20; TIN, tubulointerstitium; Mφ, macrophage; RPA, RNase protection assay; CD4, cluster of differentiation 4 (a marker of helper T-cells) ; CD8, cluster of differentiation 8 (a marker of suppressor T-cells).

EXAMPLE 2

Inhibition of Anti-GBM GN in STCl Transgenic Mice

[0093] In the following experiment, we sought to determine the impact of transgenic overexpression of STCl on inflammation, proteinuria, kidney function and blood pressure in a mouse model of anti-GBM GN. As shown in Figs IA and IB, following the administration of anti-GBM Ab, WT mice developed severe crescentic glomerulonephritis, associated with intra- and extra-capillary mononuclear cell infiltration, tubular dilatation, intra-tubular hyaline cast formation, glomerulosclerosis and interstitial fibrosis. On the other hand, STCl transgenic mice treated with anti-GBM Ab displayed 92% fewer sclerotic glomeruli, 73% fewer crescents, 75% fewer glomerular endocapillary lesions, and 82% less expansion in the tubulointerstitial compartment, compared to WT mice (Figs. IA and IB). WT mice demonstrated 3-fold increase in BUN and 50% reduction in urine output; however, there were no significant changes in BUN or urine output in STCl Tg mice (Fig. 2). In addition, STCl Tg mice exhibited less proteinuria after anti-GBM GN compared to WT mice, and importantly, while baseline blood pressure was not different between wild type and STCl Tg mice (Fig. 2), it was significantly lower in STCl tg mice after anti-GBM GN, consistent with renal protection. Control studies indicated equal deposition of sheep anti-GBM antibodies on the GBM in both, WT and Tg mice (Fig. 3A), and in agreement with a recent report (James et al, 2005), we also detect STCl in the serum of WT mice, albeit at low levels - compared with STCl Tg mice (Fig. 3B). Important for our hypothesis, transgenic mice exhibit preferential expression of STCl transgene in macrophages (Fig. 3D) and endothelial cells, where it is best appreciated in the glomerular capillaries (Fig. 4). Otherwise, the expression (Western blot on whole kidney lysate; (Fig. 3C) and distribution of STCl protein (determined by immunohistochemistry; (Fig. 4)) were similar in the kidneys of WT and STCl Tg mice. It should be emphasized that the contribution of STCl overexpression in macrophages and endothelial cells is not discernible on western blot using whole kidney lysate (Fig. 3C). Of interest, some injured tubules in WT mice (based on morphology) displayed increased expression of STCl, the significance of which remains to be determined.

EXAMPLE 3

STCl Transgenic Mice Exhibit Decreased Infiltration of the Glomeruli with Inflammatory Macrophages Following Anti-GBM GN

[0094] Macrophages and T-cells within tissue are heterogeneous: resident macrophages play an important role in tissue repair and are characterized as F4/80^hlgh, CX3CRl^hlgh, GR-l^low, CCR2^", CD62L^" and MP20^" (Chan et al, 1998; Naito, 1993; Takahashi et al, 1996) they are self-regenerative and/or may originate from circulating progenitors (Tacke and Randolph, 2006, Tacke et al., 2006) On the other hand, inflammatory/"exudative" macrophages are short-lived, do not proliferate, produce inflammatory cytokines and contribute to tissue injury; they are characterized as F4/80^low, CX3CRl^low, MP20^hlgh, _GR-l^intermediate, CCR2⁺, CD62L⁺ (Chan et al., 1998; Naito, 1993; Takahashi et al., 1996, Laskin et al., 1995). Both populations carry the pan- macrophage marker, CD68. Similarly, the nature of T-cell response (THl vs TH2) will determine cell-mediated immunity, and the course and severity of autoimmune anti-glomerular basement membrane disease (Hopfer et al., 2003).

[0095] Our published data suggest that STCl diminishes the migration of macrophages and T-cells across an endothelial monolayer (Chakraborty et al., 2006), and we hypothesized that transgenic overexpression of STCl in the endothelium and macrophages will diminish infiltration of the kidney with leukocytes, and hence, we examined the number of T-cells (CD3 ) and macrophages (total macrophage count (CD68⁺) vs resident macrophages (F4/80⁺) in the kidney, ten days after anti-GBM Ab injection - time point that correlates with peak infiltration of macrophages and T-cells in experimental mouse anti-GBM GN (Feith et al., 1996; Odobasic et al., 2005). [0096] Following anti-GBM Ab injection, resident macrophages (F4/80⁺) increased 5-fold in the interstitium and peri-glomerular region of WT mice, but only 2-fold in STCl Tg mice (Fig. 5 and Table 1). Since the number of F4/80+ macrophages in the interstitium was identical to the number of CD68⁺ macrophages (Table 1), we conclude that most, if not all interstitial macrophages in both WT and STCl Tg mice were "resident". The lower number of resident macrophages in the interstitium of STCl Tg mice compared to wild type mice is consistent with less interstitial injury in STCl Tg mice and less need for repair. Of interest, F4/80+ macrophages were nearly absent in the glomeruli of both WT and STCl Tg mice and their number did not increase after anti-GBM GN (Fig. 5 and Table 1). CD68⁺ macrophages were abundant in the glomeruli of WT mice after anti-GBM GN, but were absent in the glomeruli of STCl Tg mice after anti-GBM GN. Based on the paucity of resident macrophages (F4/80+) in the glomeruli (in both WT and STCl Tg), we conclude that most macrophages infiltrating the glomeruli of WT mice after anti-GBM GN were of the inflammatory/"exudative" variety, and these were absent in STCl Tg mice. As shown in Fig. 5 and Table 1, T-cells were predominantly interstitial and increased almost to the same degree after anti-GBM GN in both WT and STCl Tg mice (3.7- fold vs 2.7-fold, respectively). In the glomeruli, T-cells were not detected at baseline and increased minimally but equally in both WT and STCl Tg mice after anti-GBM GN.

TABLE 1

Macrophage (Mφ) and T Cell (CD3) Count in the Glomeruli and Tubulointerstitium (TIN) of WT and STCl Transgenic Mice

F4.80 is marker of resident macrophages; CD68 is a pan-macrophage marker [0097] Our data suggest that in mouse model of anti-GBM GN, inflammatory/"exudative" macrophages predominate in the glomeruli, while resident macrophages are absent; consequently, tipping the balance toward glomerular injury and less repair. Our data also suggest that circulating STCl and/or transgenic expression of STCl in macrophages and/or endothelium diminish the number of inflammatory/"exudative" macrophages within the glomeruli during anti-GBM GN, preventing kidney injury.

EXAMPLE 4

Decreased Expression of MIP-2, MCPl and TGF-β2 in the Kidney of STCl Tg Mice after Anti-GBM GN

[0098] Anti-GBM GN is associated with increased expression of several cytokines/lymphokines including IL- lβ, TNF-α, TGF-β, MIF, MIP2, MCP-I ((a) Hill et al, 1994; (b) Hill et al., 1994; Lan et al., 1997; Lan and Nikolic-Paterson et al., 1993; Neugarten et al., 1995; Yang et al., 1998; Yu et al., 1999). We performed RPA on RNA representing whole kidney tissue from WT and STCl Tg mice after anti-GBM GN and this revealed no significant changes in mRNA expression of T-cell-related cytokines (TCA-3, IL- 18, IL-6 and RANTES), and more importantly, genes characteristic of THl -mediated T-cell responses (IL12, and INFγ) or TH2-mediated responses (IL-10, GATA-3) (Fig. 6). On the other hand, MIP-2 and TGF-β2 were decreased in STCl Tg mice. The expression of MCP-I mRNA was slightly lower in STCl Tg mice, and hence, we determined protein levels using Western blotting and found reduced MCP-I protein. Additionally, we found no difference in the level of Intercellular adhesion molecule- 1 (ICAMl) protein. These data are consistent with diminished activation of inflammatory macrophages, and as a result decreased signaling to fibrosis. Our data also suggest that STCl has no significant effect on T-cell-mediated immunity in the context of anti-GBM GN, at least at the 10 day time point.

EXAMPLE 5

Decreased Deposition of Mouse C3 in the Glomeruli of STCl Transgenic Mice after Anti-GBM GN

[0099] The inflammatory injury in the acute phase of experimental anti-GBM GN is initiated by binding of the heterologous antibody to the GBM and is complement dependent (Sheerin et al., 2001). The autologous phase of the disease is mediated by the immune response against the heterologous antibody affixed to the GBM and represents a delayed hypersensitivity reaction measurable as mouse C3 and IgG deposition in the glomeruli (Le, 2004). Hence, we studied the deposition of mouse C3 and IgG in the glomeruli of WT and STCl Tg mice 10 days after the administration of sheep anti-mouse GBM Ab. While the deposition of mouse IgG in the glomeruli was similar in STCl transgenic mice and WT mice, we found little mouse C3 in the glomeruli of STCl Tg mice (Fig. 7A). Local C3 deposition and synthesis have been shown to correlate with local inflammation and cytokine release (Colten, 1992; Lappin et al, 1989; Sheerin et al., 1997). Hence, our data suggest that in spite of comparable antibody response in the autologous phase of anti-GBM GN, C3 activation is diminished in STCl Tg mice, consistent with reduced macrophage-mediated inflammation in the glomeruli. Measurements of serum IgG after anti-GBM GN in WT and STCl Tg mice revealed no differences, suggesting that STCl does not affect antibody responses (Fig. 7B).

EXAMPLE 6

Discussion of Examples 2-5

[0100] Our cumulative data suggest that stanniocalcin is a critical naturally occurring antiinflammatory protein (Kanellis et al., 2004, Chakraborty et al., 2006). It acts through a number of novel mechanisms that affect endothelial and macrophage function, when combined - these effects produce effective inhibition of inflammation. First, we found STCl decreases intracellular calcium in macrophages (Kanellis et al., 2004), and hence, is expected to diminish cell mobility, cell migration and the response to antigenic stimuli (all involve changes in intracellular calcium signal - acting as a second messenger). Indeed, we found STCl diminishes the mobility of macrophages and their response to chemoattractants (Kanellis et al., 2004). The effects of STC 1 are not limited to one chemokine, as it blocks the migration of macrophages in response to different chemokines/cytokines (Kanellis et al., 2004). Second, in addition to decreasing intracellular calcium, STCl suppresses macrophage function by decreasing superoxide generation through a novel mechanism that involves upregulation of mitochondrial uncoupling protein-2 (see below). Thirdly, the anti-inflammatory action of STCl is mediated in part through its effects on the endothelium. The endothelium plays a critical role in the migration of macrophages from the circulation to the injured tissue. Exposure of endothelial cells to cytokines (TNF-α or IL- lβ for example; produced by inflammatory cells and injured tissue) induces two key changes that facilitate leukocyte migration: opening of the tight junctions between neighboring endothelial cells, allowing inflammatory cells to traverse the endothelial barrier (Edens et al., 2000); synthesis and presentation of adhesion molecules at the cell surface, which tether circulating leukocytes to the endothelium. These changes favor the migration of inflammatory cells through the newly formed openings in the endothelial tight junctions (Edens et al., 2000; Anderson et al, 1995; Anderson et al, 1993, Dejana et al, 1995). STCl blocks the effects of cytokines (TNF-α or IL- lβ) on the endothelium, preserving endothelial permeability in cytokine-treated endothelial cells. Consistent with these data, we showed STCl dose-dependently decreases the migration of macrophages and T-cells across cytokine-treated endothelial monolayer (Chakraborty et al., 2006). The in vivo equivalent of our tissue culture results would be: 1) exposure of endothelial cells to STCl (circulating, or locally produced by the endothelium) would preserve endothelial tight junctions and inhibit extravasation of inflammatory cells to the injured tissue. 2) Exposure of macrophages to STCl decreases their mobility and function. To validate our hypothesis, we examined the outcome of anti-GBM GN (mediated by T-cells and macrophages) utilizing a transgenic mouse, which exhibits high circulating STCl protein and preferential expression of the transgene in macrophages and endothelium.

[0101] Injection of anti-GBM Ab into WT mice produced severe renal failure, associated with a 3 -fold rise in BUN and 50% reduction in urine output; on the other hand, and consistent with our hypothesis, administration of anti-GBM Ab to STCl transgenic mice produced no significant change in BUN, urine output or proteinuria, indicating protection from the injurious effects of anti-GBM Ab. This was confirmed upon review of kidney histology in STCl Tg mice, which showed no significant change in the number of sclerotic glomeruli, the number of crescentic lesions, and the degree of interstitial expansion and fibrosis after anti-GBM GN. Acute glomerulonephritis is frequently associated with an increase in blood pressure, as a result of loss of functional parenchyma, the decline in urine output, fluid retention, and activation of humoral factors (Bras et al., 1976; Rodriguez-Iturbe et al., 1981). Importantly, while baseline blood pressure was not different between wild type and STCl Tg mice, it was significantly lower in STCl Tg mice after anti-GBM GN, consistent with renal protection. [0102] A detailed analysis of the tissue provided additional insights into mechanisms of renal protection by STCl. Remarkably, the predominant macrophage population in the glomeruli of WT mice after anti-GBM GN was exudative/inflammatory (CD68⁺/F4/80^~) with almost complete absence of resident macrophages (F4/80 ). In contrast, there were no macrophages in the glomeruli of STCl Tg mice. This finding is critical and is consistent with our hypothesis, that overexpression of STCl in the endothelium should block the action of cytokines on endothelial tight junctions and preserve endothelial integrity, preventing macrophage migration across the blood vessel. Thus, attenuation of MIP-2 and MCP-I expression in the kidney and deposition of C3 in the glomeruli are consistent with inhibition of macrophage-dependent kidney injury in STCl Tg mice.

[0103] T-cells were predominantly interstitial and increased almost to the same degree in WT and STCl Tg mice after anti-GBM GN, suggesting that STCl has no significant effect on T-cell infiltration, at least at the 10-day time point. Additionally, we found no difference in the expression of cytokines characteristic of THl- or THl -mediated T-cell responses, suggesting STCl does not affect T-cell activation. Recent report suggested that macrophage depletion in the course of anti-GBM GN prevents proteinuria and glomerular macrophage infiltration, but not the accumulation of CD4⁺ or CD8⁺ T cells, indicating that macrophages are common effectors for both CD4 and CD8 T cell-dependent injury (Huang et al., 1997), and that macrophage depletion decreases the recruitment of T-cells to the injured kidney. Thus, our data are consistent with these observations and specifically implicate exudative/inflammatory macrophages in mediating kidney injury in the course of anti-GBM GN. While the number of interstitial macrophages decreased in STCl Tg mice after anti-GBM GN, compared to WT, interstitial macrophages were almost entirely of the resident variety and not inflammatory/"exudative"; and the smaller number of interstitial resident macrophages in STC 1 Tg after anti-GBM GN may be a reflection of lesser injury, and hence, lesser need for reparative macrophages. Alternatively, STCl may have direct effect to decrease interstitial macrophage proliferation or recruitment. [0104] Paciga, McCudden and their colleagues reported the existence of STCl -binding protein/receptor in multiple tissues (McCudden et al., 2002; Paciga et al., 2003; McCudden et al., 2004). They used a stanniocalcin-alkaline phosphatase fusion protein in binding assays and concluded there are high-affinity (0.25-0.8 nM), saturable and displaceable binding sites for STCl (McCudden et al., 2002; Paciga et al., 2003; McCudden et al., 2004). Besides impairing macrophage function, it's unknown if STCl affects the function of other cells involved in inflammation. In conclusion, STCl effectively blocks inflammation in the kidney.

EXAMPLE 7

Experimental Procedures for Examples 8-14

[0105] Materials: All materials were purchased from Sigma (St Louis, MO) unless stated otherwise. Recombinant hSTCl protein was kindly provided by Dr. Henrik Olsen, Human Genome Sciences (Rockville, MD). It was expressed in a baculovirus expression system and is greater than 90% pure (Zhang et al., 1998). Endotoxin levels in STCl preparation were determined using Limulus Amebocyte Lysate Test Kit (Cambrex Bio Science Walkersville, MD) according to manufacturer's instructions, and showed no detectable endotoxin. Goat anti- UCP2 antibodies were purchased from Santa-Cruz (Santa Cruz, CA).

[0106] Preparation of peritoneal macrophages: peritoneal macrophages were harvested from male wild type and UCP ^" mice (The Jackson Laboratory, Bar Harbor, ME); both have C57B/6 genetic background. Three days after injecting 3% Brewer's thioglycolate into the peritoneal cavity, cells were harvested by irrigation of the peritoneal cavity with sterile saline solution (0.9% NaCl). The collected cells were centrifuged and the cell pellet was suspended in Dulbecco's Modified Eagle's Medium (DMEM). Cells were allowed to adhere on 6-well culture plates for 2 h at 37° C with 5% CO₂. Non-adherent cells were removed by washing with PBS and the remaining adherent cells were suspended in DMEM containing 10% fetal bovine serum (FBS).

[0107] Measurement of cellular ATP level: Freshly-isolated peritoneal or cultured murine macrophages (RAW264.7) were maintained in DMEM supplemented with 10% FBS, and cellular ATP content was measured using bioluminescent somatic cell ATP assay kit (Sigma), as per manufacturer's instructions. Briefly, cells were washed in ice-cold PBS and lysed in somatic cellular ATP releasing reagent. Cell lysates were incubated with ATP assay mix, and cellular ATP levels were measured as bioluminescence using a TD-20/20 luminometer (Turner Designs Instruments, Sunnyvale, CA), and data were expressed as % of controls.

[0108] Mitochondrial respiratory chain activity assay: Cell homogenates were prepared by sonication in a buffer containing 250 mM sucrose, 2 mM EDTA and 100 mM Tris-HCl, pH 7.4 (Wiedemann et al., 2002). The assay was carried out on whole cell lysate at 30° C using a temperature-controlled spectrophotometer (Pharmacia, Biotech; Piscataway, NJ). The activities of mitochondrial respiratory chain complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex I + III (NADH cytochrome c reductase), complex II + III (succinate:cytochrome c reductase) and complex IV (cytochrome c oxidase) were assayed using different electron acceptors/donors as previously described (Sottocasa et al., 1967; Vu et al., 1998). NADH dehydrogenase activity was measured as the rate of NADH oxidation (measurement of NADH absorbance at 340 nm), using potassium ferricyanide as the electron acceptor. Succinate dehydrogenase activity was measured as the rate of 2,6- dichlorophenolindophenol (DCIP) reduction (measurement of DCIP absorbance at 600 nm), using succinate as electron donor (reaction is carried out in the presence of KCN - to inhibit cytochrome c oxidase). NADH cytochrome c reductase activities were measured as the rate of cytochrome c reduction (measurement of cytochrome c absorbance at 550 nm), using NADH as electron donor (reaction is carried out in the presence of KCN - to inhibit cytochrome c oxidase). The activities of succinate:cytochrome c reductase were measured as the rate of cytochrome c reduction (measurement of cytochrome c absorbance at 550 nm), using succinate as electron donor (reaction is carried out in the presence of KCN - to inhibit cytochrome c oxidase). Cytochrome c oxidase activity (measurement of cytochrome c absorbance at 550 nm) was measured as the rate of oxidation of freshly reduced cytochrome c, using Na hydrosulfate. To adjust enzymatic activities for mitochondrial content, the activities were expressed as percentage of values in controls and normalized to citrate synthase activity - measured as the reaction of sodium oxaloacetate, acetyl-coenzyme A and 5,5'-dithio-bis-(2 nitrobenzoic) acid at 412 nm (Sherratt et al, 1988).

[0109] Western blot analysis: macrophages were treated with hSTCl (100 ng/ml) or vehicle for varying times, and lysed in modified RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, containing 1 mM phenylmethylsulfonylfluoride and 1 μg/ml leupeptin) and centrifuged for 10 min at 1400 rpm/4° C to remove cell debris. Fifty μg of total cell lysates were resolved on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane and incubated with goat anti-UCP2 antibody (Santa-Cruz, CA). After washing with TBST buffer (20 mM Tris, pH 7.6, 137 mM NaCl, 0.1% Tween-20), the membrane was incubated with horseradish peroxidase-conjugated anti-goat IgG. The bound antibodies were visualized using chemiluminescence.

[0110] Analysis of cell cycle and apoptosis: cultured murine macrophages were plated in DMEM containing 10% FBS, 1% penicillin/streptomycin and grown in humidified incubator at 37° C in 5% C(V95% air. At the start of the experiment, medium was replaced with DMEM containing 1% FBS. In some experiments, cells were maintained in DMEM + 10% FBS. Cells were then treated with hSTCl (100 ng/ml) or vehicle, while suspended in DMEM plus 10% FBS or DMEM plus 1% FBS. After treatment, cells were rinsed with PBS, fixed with 70% ethanol, stained with propidium iodide and treated with RNase according to established protocol (Wang et al., 2002). Cells were analyzed using fluorescence-activated cell sorter (FACStar flow cytometer; BD Biosciences, San Jose, CA).

[0111] Measurement of mitochondrial membrane potential: Mitochondrial membrane potential was measured using 3,3'-tetraethylbenzimidazolylcarbo-cyanine iodide (JC-I), as per manufacturer's instructions (Molecular Probe, Eugene, OR). JC-I is a cationic dye that indicates mitochondrial polarization by shifting its fluorescence emission from green (-530 nm) to red (-590 nm). Cultured murine macrophages were seeded overnight then treated with hSTCl protein for the indicated times. After treatment, cells were incubated in 50 nM JC-I at 37° C for 30 min and washed (3X) with PBS. The red (mitochondria) JC-I fluorescence was measured at 530 nm excitation/590 nm emission, and the green (cytoplasmic) JC-I fluorescence was measured at 485 nm excitation/530 nm emission using a fluorescence-activated fluorescence reader (BMG Labtechnologies, Germany). After subtraction of background values obtained from wells containing JC-I but devoid of cells, red/green fluorescence ratios were calculated. [0112] LDH assay: Leakage of lactate dehydrogenase (LDH) from the cells was measured as an index of lethal membrane injury (necrosis). At the end of the experiment, cell culture medium was collected, and LDH was measured using LDH assay kit (Sigma) according to manufacturer's protocol. LDH values were expressed as percent of controls. [0113] Superoxide measurement: Macrophages were treated with or without STCl for 6h or 24h. During the last 15 min of treatment, cells were incubated with 10 μM dihydroethidium (DHE). After wash with ice-cold PBS, the cells were harvested and suspended in PBS on ice. The percentage of ethidium bromide positive cells was determined by flow cytometry. [0114] Statistical analysis: Data were expressed as mean ±SEM. Statistical significance was determined by student t test. Ap value of <0.05 was considered statistically significant. [0115] Nonstandard abbreviations used: STCl, stanniocalcin- 1 ; UCP, uncoupling protein; LDH, lactic dehydrogenase; NADH, Nicotinamide adenine dinucleotide; DCIP, 2,6- dichlorophenolindophenol; JC-I, 3,3'-tetraethylbenzimidazolylcarbo-cyanine iodide; DHE, dihydroethidium; ROS, reactive oxygen species; LPS, Lipopolysacharide.

EXAMPLE 8

STC 1 Decreases Total Cellular ATP Content in Cultured Murine Macrophages [0116] We determined the effect of STCl on cellular ATP level in freshly-isolated peritoneal macrophages and cultured murine macrophages. STCl decreased total cellular ATP content in a dose- and time-dependent manner. As shown in Fig. 8A, after Ih incubation with STCl, maximal attenuation of ATP level (60%) was observed at 500 ng/ml STCl (IC50 of 50 ng/ml). At an intermediate concentration of STCl (100 ng/ml), which have been reported to exist in vivo in various organs (De Niu et al, 2000), we observed 50% reduction in ATP levels after 3 hours (Fig. 8B). Of interest, the decline in ATP level was sustained for 24 hours after a single treatment with STCl, however, washout experiment suggested that the effects of STCl are reversible (Fig. 8C). However, denatured STCl had no effect on cellular ATP levels (Fig. 8D). Of interest, we observe a marked increase in intracellular labeling for STCl 10 min after the addition of recombinant STCl to the medium and localization to the mitochondria, suggesting internalization of STCl by macrophages and, hence, an important role for circulating or free tissue STCl in regulating macrophage function (Fig. 8E). The data suggest that STCl plays an important role in regulating energy metabolism in macrophages and possibly in other cells and organs.

EXAMPLE 9

STCl Does not Affect the Activity of Mitochondrial Respiratory Chain Complexes I-IV [0117] Since oxidative phosphorylation is the main source of cellular ATP production, we investigated whether STCl affects the activity of respiratory chain proteins in cultured murine macrophages. Using total cell lysates we found no differences in the activities of complex I, complex II, complex I+II, complex II+III, and complex IV in STCl -treated cells compared with vehicle-treated controls (Fig. 9). These data rule out inhibition of mitochondrial respiratory chain complexes as the cause for the decline in intracellular ATP levels in STCl -treated cells.

FIGURE 10

STCl Induces UCP2 Protein Expression in Macrophages

[0118] Uncoupling proteins (UCPs) are mitochondrial anion carriers, that localize at the inner mitochondrial membrane and facilitate H⁺ "leak" from the mitochondrial inter-membrane space to the matrix, diminishing H⁺ gradient which drives ATP generation (Rousset et al., 2004). UCP2 is highly expressed in the lymphoid system and macrophages (Nishio et al., 2005), and plays an important role in superoxide generation and macrophage activation (Nishio et al., 2005, Bai et al., 2005; Arsenijevic et al., 2000). In the following experiment, we examined the effect of STCl on UCP2 expression in cultured murine macrophages and found time-dependent increase in UCP2 protein expression (Fig. 10). The increase in UCP2 protein occurs within the first hour after incubation with STCl and is 3-fold higher after 3-4 h. UCP2 is regulated predominantly at the translational level (Hurtaud et al., 2006), and immediate downregulation of UCP2 protein expression in macrophages have been reported to occur within the first hour after treatment with Lipopolysacharide (LPS) (Emre et al., 2007). Thus, the immediate increase in UCP2 in response to STCl is consistent with UCP2 gene responses. We found no expression of UCP3 in cultured macrophages (data not shown), confirming previously published data (Ishioka et al., 2002). Since STCl upregulates UCP2 in macrophages, while LPS downregulates UCP2 - to increase mitochondrial superoxide generation (Emre et al., 2007), our data also suggest a potential role for STCl as an endogenous LPS antagonist. Our data identify STCl as an inducer of UCP2 in macrophages and suggest an important role for STCl in the regulation of superoxide generation in macrophages.

EXAMPLE 11

STCl Attenuates Mitochondrial Membrane Potential in Macrophages

[0119] Upregulation of UCP2 is expected to shunt protons from the mitochondrial inter- membrane space to the matrix and decrease hydrogen ion gradient between the mitochondrial inter-membrane space and matrix. A decline in H⁺ ion gradient is expected to reduce mitochondrial membrane potential (depolarization), but the correlation between the two is not linear; for example, transient uncoupling of mitochondrial oxidative phosphorylation using pharmacologic uncouplers results in modest depolarization of mitochondrial membrane potential; however, this modest depolarization is sufficient to decrease the generation of reactive oxygen species (Dimroth et al., 2000; Sack 2006). Using JC-I, a fluorescent indicator of mitochondrial membrane potential, we found progressive depolarization of the mitochondria in STCl -treated cells (Fig. 11). Membrane potential was 20% lower at 5h and nearly 60% lower after 24h incubation with STCl (150 ng/ml). These data are consistent with STCl -induced and UCP2-mediated dissipation of H⁺ gradient between the mitochondrial inter-membrane space and matrix, and predict attenuation of superoxide generation.

EXAMPLE 12

STCl -Induced Reduction in Superoxide Generation is UCP2-Dependent

[0120] Since uncoupling of mitochondrial oxidative physphorylation is expected to decrease the generation of reactive oxygen species (Arsenijevic et al., 2000; Horimoto et al., 2004), we then determined whether STCl inhibited superoxide generation. As shown in Fig. 12, treatment of freshly isolated peritoneal macrophages from wild type mice with STCl for 24h led to 40% reduction in superoxide generation, as determined by dihydroethidium (DHE) staining. However, treatment of peritoneal macrophages from UCP2^7" mice with STC 1 had no effect on superoxide generation. We found no changes in the expression of gp91phox and p22phox subunits of NADPH oxidase in STCl -treated macrophages (data not shown), suggesting STCl has no effect on NADPH oxidase. These data suggest that STCl -induced reduction in superoxide generation is UCP2-dependent and that STCl may have profound effects on macrophage function and viability.

EXAMPLE 13

STCl Induces Cell Cycle Arrest at the Gl Phase in Macrophages

[0121] The decline in cellular ATP levels can lead to cell death (Saikumar et al., 1998). On the other hand reactive oxygen species are known to induce apoptosis (Kubota et al., 2005; Madesh et al., 2005), and hence, the decline in superoxide generation in STCl-treated macrophages is expected to improve cell viability. In the following experiments, we sought to determine the effect of STCl on necrosis and apoptosis in serum starved cultured murine macrophages: these were measured based on LDH release to the medium and cellular DNA content (determined by FACS), respectively; both methods are widely used for the assessment of cell viability. As shown in Fig. 13A, STCl resulted in 60% reduction in LDH leak from serum-starved cells, consistent with decreased necrosis in response to serum starvation. Similarly the fraction of apoptotic cells was lower in STCl-treated cells compared to controls (5% vs 26%, respectively); analysis of DNA content suggested that STCl treatment arrested a large proportion of the cells at the Gl phase (71% of STCl-treated cells, vs 51% in controls; (Fig. 13B). Qualitatively similar results were observed when cells were maintained in DMEM + 10% fetal bovine serum (data not shown). Our data suggest that in spite of the decline in ATP levels in STCl-treated cells, cell viability is improved, and this survival advantage may result from the reduction in reactive oxygen species. We also speculate that the arrest at the Gl phase may have spared the cells from proceeding to apoptosis.

EXAMPLE 14 Discussion of Examples 8-13

[0122] As discussed earlier, STCl functions as a true hormone in bony fish, where it is produced by one organ - the gland of Stannius, and acts in the gut and the gill, where it diminishes calcium influx from the aquatic environment to the blood. Mammalian STCl is ubiquitously expressed (Chang et al., 1995; Olsen et al., 1996; Varghese et al., 1998) and circulates in the blood (James et al., 2005); hence, it may have hormonal as well as autocrine/paracrine functions. The function of mammalian STCl is not fully defined. Previous data from our laboratory suggested that STCl is an anti-inflammatory protein. This conclusion was based on the following observations: In macrophages, STCl attenuates intracellular second messenger signals through calcium (Kanellis et al, 2004); it diminishes the response of macrophages to chemokines (Kanellis et al., 2004), and decreases transendothelial migration of human macrophages and T-lymphocytes (Chakraborty et al., 2006).

[0123] Our current results show that STCl treatment induces UCP2 in macrophages. It decreases both mitochondrial membrane potential and superoxide generation. This is relevant to immunity and inflammation because mitochondria generate superoxides (O₂), which are critical for macrophage function. Transfer of electrons (e ) from high energy molecules (NADH & FADH2) to the respiratory chain (complexes I-IV) generates a higher H⁺ ion concentration in the mitochondrial inter-membrane space compared to matrix. These reactions also produce superoxide (primarily through the actions of complexes I and III). H⁺ ions are allowed back to the matrix through the ATP synthase protein complex, facilitated by the favorable electrochemical gradient for H⁺; the H⁺ movement in turn drives the generation of ATP (Wada et al., 2000; Schultz et al., 2001; Jain et al., 2000). Alternatively, the H⁺ gradient may be dissipated by uncoupling proteins (such as UCP2); these proteins localize to the inner mitochondrial membrane and introduce a channel which allows H⁺ ion "leak" from the inter- membrane space back into the matrix, bypassing ATP synthase. This response to uncoupling proteins diminishes both ATP synthesis and superoxide generation (Rousset et al., 2004; Brand et al., 2005, Brand et al., 2004).

[0124] The best known uncoupling protein is UCPl, which is expressed in brown adipocytes; UCP 1 uncouples ATP generation from electron transport, and the uncoupling process enhances respiration, producing a futile cycle which dissipates oxidative energy as heat (thermoregulation; Thompson et al., 2004). UCP3 (muscle), UCP4 and UCP5 (brain), limit free radical production (Brand et al., 2005; Kim-Han et al., 2005). UCP2 is highly expressed in lymphoid cells including macrophages (Nishio et al., 2005; Arsenijevic et al. (Arsenijevic et al., 2000), and there is increasing evidence for the involvement of UCP2 in immunity. Arsenijevic et al., 2000 reported that UCP2^7" mice were resistant to infection by Toxoplasma gondii; macrophages from UCP2^7" mice generated 80% more reactive oxygen species (ROS) than macrophages from wild- type mice in response to Toxoplasma gondii, and had fivefold greater toxoplasmacidal activity in vitro. Notably, the benefits of UCP2^7" in macrophages were abolished by a quencher of ROS (Arsenijevic et al., 2000). Similarly, a recent report by Bai et al. (Bai et al., 2005) showed that disruption of UCP2 gene was associated with greater free radical production in macrophages, enhanced macrophage phagocytic activity and resistance to infectious microorganisms. Mitochondrially-generated ROS in UCP2^"7" macrophages activate NF-κB, resulting in a "primed state", which potentiates and amplifies subsequent inflammatory responses (Bai et al, 2005); activation of NF-κB in turn, stimulates the expression of chemokines, immune receptors, adhesion molecules and cell cycle regulators (Tergaonkar 2006). In contrast, transfection of macrophages with UCP2-expressing vector dissipates mitochondrial membrane potential reducing ROS production and NF-κB activation, diminishing the expression of downstream gene targets such as cytokines (Nishio et al., 2005; Bai et al., 2005). Of interest, the proinflammatory effects attributed to Lipopolysaccharide (LPS) are mediated in part through suppression of UCP2 (Emre et al., 2007), and as a result, increased mitochondrial superoxide generation. These data suggest that mitochondrial superoxide generation is critical for macrophage function and that UCP2 is central to the regulation of this superoxide source. Importantly, STCl emerges as a key regulator of UCP2 and superoxide generation in macrophages and may play an important role in opposing the effects of LPS, and hence may be important in regulating innate immunity.

[0125] As discussed earlier, STCl is ubiquitously expressed, and localizes to many tissues including the brain, heart, skeletal muscle, ovaries, testes, kidneys, pancreas, spleen and adipocytes; similarly, UCPs are widely expressed and parallel STCl in their distribution (Ishioka et al., 2002; Kim-Han et al., 2005, Digby et al., 2000, Lentes et al., 1999). Thus, it is tempting to speculate that STCl may function as a regulator of UCPs in other tissues and organs. Hence, we propose that in addition to reducing reactive oxygen species and enhancing cell survival in macrophages, STCl may induce uncoupling proteins in other tissues and have a role in regulating superoxide generation outside the lymphatic system. This hypothesis is supported by previous observations in STCl-overexpressing mice, which were hyperphagic (Filvaroff et al., 2002), displayed enlarged mitochondria, had leaner fat pads and faster clearance of glucose from the circulation (Filvaroff et al., 2002); yet they were 40% smaller compared to age-matched wild-type litter-mates (Filvaroff et al., 2002; Varghese et al., 2002). This is interesting because overexpression of UCP3 in skeletal muscle produces hyperphagic mice that are smaller in size compared to wild type litter-mates and display leaner fat pads and faster clearance of glucose (Clapham et al., 2000), a phenotype that closely resembles that of STCl overexpressing mice (Filvaroff et al., 2002; Varghese et al., 2002).

[0126] Lastly, treatment of macrophages with STCl attenuates cell necrosis and apoptosis, and induces cell cycle arrest at the Gl phase. The improvement in cell survival may result from the decrease in superoxide generation, while the arrest at the Gl phase may be linked to the decline in cellular ATP levels. Consistent with that, a recent report suggested that lower cellular ATP levels may promote cell survival by inducing cell cycle arrest (Mandal et al., 2005). [0127] The key question remains, does STCl provide anti-inflammatory action. In Examples 1-6 above we examined the effect of transgenic overexpression of STCl on inflammation, using anti-glomerular basement membrane disease, a model of rapidly progressive glomerulonephritis, and is characterized by proteinuria, macrophage and T-cell infiltration, glomerular crescent formation. Macrophages and T-cells play a critical role in the pathogenesis of anti-GBM glomerulonephritis, and their number correlates with the percentage of crescentic glomeruli (Hattori et al., 1994; Coelho et al., 1997; Wu et al., 2002; Hopfer et al., 2003; Huang et al., 1997, Huang et al., 1997, Tipping et al., 1985). Transgenic overexpression of STCl diminishes cytokine activation, inhibits infiltration of the glomeruli with inflammatory macrophages and provides renal protection from anti-glomerular basement membrane disease. Thus, STCl is a critical endogenous anti-inflammatory protein.

EXAMPLE 15

Stanniocalcin-1 Suppresses Superoxide Generation in Cardiomyocytes

[0128] STCl transgenic mice: Mice expressing hSTCl under the control of metallothionein I minimal promoter as described above were utilized in this Example. Animal manipulations were conducted in accordance with national and institutional guidelines.

[0129] Measurement of cellular ATP level: Cellular ATP content was measured using bioluminescent cell ATP assay kit (Sigma). Briefly, hearts were homogenized in somatic cellular ATP releasing reagent, and lysates were incubated with ATP assay mix. ATP was measured as luminescence, and expressed as % of the WT.

[0130] Mitochondrial respiratory chain activity assay: NADH dehydrogenase (complex I) was measured as the oxidation of NADH using potassium ferricyanide as the electron acceptor. Succinate dehydrogenase (complex II) was measured as the reduction of 2,6- dichlorophenolindophenol. NADH cytochrome c reductase (complex I + III) were measured as the reduction of cytochrome c using NADH as electron donor. Succinate cytochrome c reductase (complex II + III) were measured as the reduction of cytochrome c using succinate as electron donor. Cytochrome c oxidase (complex IV) was measured as oxidation of dithionite-reduced cytochrome c. All assays were performed spectrophotometrically at 30⁰C. The activities were expressed as % of WT and normalized to citrate synthase activity, measured as the reaction of sodium oxaloacetate, acetyl-coenzyme A and 5,5'-dithio-bis-(2 nitrobenzoic) acid at 412 nm (71 from manuscript T).

[0131] Cardiomyocyte preparation: one week old adult rat cardiomyocytes (Sheikh-Hamad et al., 2003) were treated overnight with STCl (100 ng/ml). Superoxide was measured using the DHE method as described above.

[0132] As shown in the preceding Examples, STCl induces uncoupling protein 2 and attenuates mitochondrial membrane potential thereby decreasing intracellular ATP and superoxide generation in macrophages. We hypothesized that STCl may affect energy metabolism/superoxide generation in the heart. In the current Example, we examined the impact of transgenic overexpression of STCl on cardiac: 1) ATP levels; 2) the activity of mitochondrial respiratory chain complexes (I-IV); 3) expression of uncoupling proteins 2 and 3 (UCP2 and 3). In addition, we examined the effect of recombinant STCl on angiotensin II-provoked superoxide generation and UCP3 expression in cultured rat cardiomyocytes. As shown in Fig. 14, ATP levels in cardiac tissue of STCl Tg mice were 60% lower, compared with WT litter mates. As shown in Fig. 15, the activities of respiratory chain complexes I-IV in heart tissue lysates of Tg mice were similar to WT mice; however, UCP3 protein levels were elevated in STCl Tg mice. In cultured rat cardiomyocytes, STCl induced UCP3 protein expression (Fig. 16) and suppressed angiotensin II-mediated superoxide generation (Fig. 17). Thus, upregulation of STCl in cardiomyopathy diminishes superoxide generation and provides cardioprotection.

EXAMPLE 16

Stanniocalcin- 1 is a Naturally Occurring Anti-Oxidant That Can Function as Cardioprotectant in Ischemic or Hypertrophic Cardiomyopathies

[0133] Mice: C57BL/6 mice were used. Animal manipulations were conducted in accordance with national and institutional guidelines.

[0134] Aortic banding protocol: Male and female, 12-20 weeks old mice were anesthetized by an IP injection of pentobarbital (60 μg/g). Aortic banding was achieved by creating a constriction between the right innominate and left carotid arteries. A 6-0 suture was tied twice around a blunt 3-mm segment of a 27-gauge needle, which was positioned adjacent to the aorta and was removed after placement of the ligature. Pressure overload was measured by right-to- left carotid artery flow velocity ratio after constricting the transverse aorta. Only mice with a flow ratio from 5: 1 to 10: 1 were used for analysis. As a control, a "sham" operation without aortic constriction was performed on age-matched mice. At the end of the experiment, the heart was excised, fixed in zinc- formalin, and embedded in paraffin, or frozen. [0135] Cardiac ischemia protocol: Male and female, 8-12 weeks old mice were anesthetized by an IP injection of pentobarbital (60 μg/g). A closed-chest mouse model of reperfused myocardial infarction was used to avoid the confounding effects of surgical trauma and inflammation. The LAD was occluded for lhr then reperfused for 3, 7 or 28 days. Sham animals were prepared identically without undergoing coronary occlusion/reperfusion. At the end of the experiment, the heart was excised, fixed in zinc-formalin, and embedded in paraffin, or snap- frozen and stored at -80° C.

[0136] Cardiomyocyte preparation: One week old adult rat cardiomyocytes (Sheikh-Hamad et al., 2003) were treated overnight with STCl (100 ng/ml). Superoxide was measured using DHE method as described above.

[0137] SDS-PAGE and mass spectrometry: Total heart lysates were resolved on 12% SDS PAGE, transferred to nitrocellulose membrane and incubated with rabbit anti- STCl antibody (Santa-Cruz). The bound antibodies were visualized using chemiluminescence. Band intensities were normalized to actin.

[0138] As shown in the preceding Examples, STCl suppresses superoxide generation in macrophages through induction of mitochondrial uncoupling protein-2 (UCP2). STCl expression in cardiomyocytes is IL6-dependent; while angiotensin II (A II) induces a number of cytokines including IL-6. We hypothesized that STCl expression in the heart will be increased in experimental settings where All is increased. In this Example, we examined STCl expression in the heart of mice after aortic banding, a model of angiotensin-II-mediated hypertension and cardiac hypertrophy; and after coronary ligation, a model of ischemic cardiomyopathy. Additionally, using STCl -treated cultured primary adult rat cardiomyocytes, we sought to determine: A) the expression of UCP3, the prevailing uncoupling protein in cardiomyocytes; B) angiotensin II-mediated superoxide generation. As shown in Figs. 18 and 20 aortic banding (for 3 days, 7 days or 28 days) led to an increase in the expression of STCl in the heart. In addition, aortic banding led to an increase in the expression of UCP3 in the heart (Fig. 19). Labelling is predominantly peripheral. STCl and UCP3 show parallel expression and distribution. Similarly, cardiac expression of STCl was increased in the ischemia model (Fig. 21). STCl induced UCP3 in cultured cardiomyocytes (Fig. 22), and it suppressed angiotensin II-mediated superoxide generation (Fig. 22). This Example demonstrates that that: 1) Cardiac expression of STCl is increased in models of ischemic and hypertrophic cardiomyopathies, where angiotensin II is typically high and 2) STCl suppresses angiotensin II-mediated superoxide generation in cultured cardiomyocytes. This data suggest that STCl functions as a naturally occurring antioxidant and cardioprotectant in ischemic or hypertrophic cardiomyopathies.

[0139] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

[0140] It will be appreciated that the methods and compositions of the instant invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. BIBLIOGRAPHY

[0141] Anderson, J.M. et al. (1993). The structure and regulation of tight junctions. Curr Opin Cell Biol 5:772-778.

[0142] Anderson, J.M. et al. (1995). Tight junctions and the molecular basis for regulation of paracellular permeability. Am J Physiol 269:G467-G475.

[0143] Anwer, K. et al. (2000). Ultrasound enhancement of cationic lipid-mediated gene transfer to primary tumors following systemic administration. Gene Ther 7: 1833-1839.

[0144] Arsenijevic, D. et al. (2000). Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat Genet 26:435-439.

[0145] Bai, Y. et al. (2005). Persistent nuclear factor-kappa B activation in Ucp2-/- mice leads to enhanced nitric oxide and inflammatory cytokine production. J Biol Chem 280: 19062- 19069.

[0146] Bolli, R. et al. (1995). Recurrent ischemia in the canine heart causes recurrent bursts of free radical production that have a cumulative effect on contractile function. A pathophysiological basis for chronic myocardial "stunning". J Clin Invest 96: 1066-1084.

[0147] Brand, M.D. et al. (2004). Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins. Free Radio Biol Med 37:755-767.

[0148] Brand, M.D. et al. (2005). Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3. Cell Metab 2:85-93.

[0149] Bras, H. et al. (1976). Plasma renin activity (PRA) and aldosterone (PA) in patients with chronic glomerulonephritis (GN) and hypertension. Clin Nephrol 5:57-60.

[0150] Cesselli, D. et al. (2001). Oxidative stress-mediated cardiac cell death is a major determinant of ventricular dysfunction and failure in dog dilated cardiomyopathy. Circ Res 89 279-286.

[0151] Chakraborty, A. et al. (2007). Stanniocalcin- 1 regulates endothelial gene expression and modulates trans-endothelial migration of leukocytes. Am J Physiol Renal Physiol 292:F895- 904.

[0152] Chang, A.C. et al. (1995). A novel human cDNA highly homologous to the fish hormone stanniocalcin. MoI Cell Endocrinol 112:241-247.

[0153] Chang, A.C. et al. (2003). Mammalian stanniocalcins and cancer. Endocr Relat Cancer 10:359-373.

[0154] Clapham, J.C. et al. (1992). Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean. Nature 406:415-418, 2000 Colten HR: Tissue-specific regulation of inflammation. JAppl Physiol 72: 1-7 ^'.

[0155] Cleland, J.L. et al. (2001). Emerging protein delivery methods. Curr Opin Biotechnol 12:212-219.

[0156] Coelho, S.N. et al. (1997). Immunologic determinants of susceptibility to experimental glomerulonephritis: role of cellular immunity. Kidney Int 51 :646-652.

[0157] Dejana E. et al. (1995). Endothelial cell-to-cell junctions. FASEB J9:910-918, 1995 [0158] De Niu, P. et al. (2000). Development of a human stanniocalcin radioimmunoassay: serum and tissue hormone levels and pharmacokinetics in the rat. MoI Cell Endocrinol 162: 131- 144.

[0159] Digby, J.E. et al. (2000). Depot-related and thiazolidinedione-responsive expression of uncoupling protein 2 (UCP2) in human adipocytes. Int J Obes Relat Metab Disord 24:585- 592.

[0160] Dimroth, P. et al. (2000). Crucial role of the membrane potential for ATP synthesis by F(I)F(O) ATP synthases. J Exp Biol 203:51-59.

[0161] Edens, H.A. et al. (2000). Modulation of epithelial and endothelial paracellular permeability by leukocytes. Adv Drug Deliv Rev 41:315-328.

[0162] Emre, Y. et al. (2007). Mitochondria contribute to LPS-induced MAPK activation via uncoupling protein UCP2 in macrophages. Biochem J402:271-278.

[0163] Feith, G. W. et al. (1996). Different mediator systems in biphasic heterologous phase of anti-GBM nephritis in mice. Nephrol Dial Transplant 11:599-607.

[0164] Filvaroff, E.H. et al. (2002). Stanniocalcin 1 alters muscle and bone structure and function in transgenic mice. Endocrinology 143:3681-3690.

[0165] Flaherty, J.T. and Weisfeldt, MX. (1988). Reperfusion injury. Free Radic Biol Med 5:409-419.

[0166] Fontaine, M. (1964). Corpuscles de Stannius et regulation ionique (Ca, K, Na) du Milieu interieur de l'Anguille (Anguilla anguilla L.). CR Acad Sc Paris Serie D 259:875-878.

[0167] Greenwald, R.B. et al. (2003). Effective drug delivery by PEGylated drug conjugates. Adv Drug Deliv Rev 55:217-250.

[0168] Hammerland, L.G. et al. (1992). Conantokin-G selectively inhibits N-methyl-D- aspartate-induced currents in Xenopus oocytes injected with mouse brain mRNA. Eur. J. Pharmacol. 226:239 '-244.

[0169] Harris, J.M. and Chess, R.B. (2003). Effect of pegylation on pharmaceuticals Nat Rev Drug Discov 2:214-221.

[0170] Hattori, T. (1994). Contribution of ED-I- and CD-8-positive cells to the development of crescentic-type anti-GBM nephritis in rats. Nippon Jinzo Gakkai Shi 36: 1228-1239.

[0171] Hill, P.A. et al. (1994). The ICAM-l/LFA-1 interaction in glomerular leukocytic accumulation in anti-GBM glomerulonephritis. Kidney Int 45:700-708.

[0172] Hill, P.A. et al. (1994). ICAM-I directs migration and localization of interstitial leukocytes in experimental glomerulonephritis. Kidney Int 45:32-42, 1994

[0173] Hopfer, H. et al. (2003). The importance of cell-mediated immunity in the course and severity of autoimmune anti-glomerular basement membrane disease in mice. FASEB J 17:860- 868.

[0174] Horimoto, M. et al. (2004). Uncoupling protein-2 deficiency promotes oxidant stress and delays liver regeneration in mice. Hepatology 39:386-392.

[0175] Huang, X.R. et al. (1997). Mechanisms of T cell-induced glomerular injury in anti- glomerular basement membrane (GBM) glomerulonephritis in rats. Clin Exp Immunol 109: 134- 142. [0176] Hurtaud, C. et al. (2006). Translation control of UCP2 synthesis by the upstream open reading frame. Cell MoI Life Sci 63: 1780-1789.

[0177] Ishioka, K et al. (2002). Canine mitochondrial uncoupling proteins: structure and mRNA expression of three isoforms in adult beagles. Comp Biochem Physiol B Biochem MoI Biol 131 :483-489.

[0178] Iyer, V.R. et al. (1999). The transcriptional program in the response of human fibroblasts to serum. Science 283:83-87.

[0179] Jain, S. et al. (2000). Kinetic model of ATP synthase: pH dependence of the rate of ATP synthesis. FEBS Lett 476: 113-117.

[0180] James, K. et al. (2005). Evidence for stanniocalcin binding activity in mammalian blood and glomerular filtrate. Kidney Int βl:All-A%2.

[0181] Kahn, J. et al. (2000). Gene expression profiling in an in vitro model of angiogenesis. Am J Pathol 156: 1887-1900.

[0182] Kanellis, J. et al. (2004). Stanniocalcin- 1, an inhibitor of macrophage chemotaxis and chemokinesis. Am J Physiol Renal Physiol 286:F356-362.

[0183] Kim-Han, J.S. et al. (2005). Mitochondrial uncoupling proteins in the central nervous system. Antioxid Redox Signal 7: 1173-1181.

[0184] Kodera, Y. et al. (1998). Pegylation of proteins and bioactive substances for medical and technical applications. Prog in Polymer Sci 23 : 1233-1271.

[0185] Kubota, Y. et al. (2005). Radiation-induced apoptosis in peritoneal resident macrophages of C3H mice: selective involvement of superoxide anion, but not other reactive oxygen species. Int J Radiat Biol 81 :459-472.

[0186] Lafeber, F. P. et al. (1988). Hypocalcin from Stannius corpuscles inhibits gill calcium uptake in trout. Am J Physiol Renal Fluid Electrolyte Physiol 254:F891-F896.

[0187] Lan, H.Y. et al. (1997). The pathogenic role of macrophage migration inhibitory factor in immunologically induced kidney disease in the rat. J Exp Med 185: 1455-1465.

[0188] Lan, H.Y. et al. (1993). Suppression of experimental crescentic glomerulonephritis by the interleukin-1 receptor antagonist. Kidney Int 43:479-485.

[0189] Lappin, D. et al. (1989). Modulation of monocyte complement synthesis by lymphocytes and lymphocyte-conditioned media. Clin Exp Immunol 76:86-91.

[0190] Laskin, D. L. et al. (1995). Macrophages and inflammatory mediators in tissue injury. Annu Rev Pharmacol Toxicol 35:655-677.

[0191] Lawrie, A. et al. (2000). Microbubble-enhanced ultrasound for vascular gene delivery. Gene Ther 7:2023-2027.

[0192] Le, H.M. et al. (2004). Histopathology of humorally mediated anti-glomerular basement membrane (GBM) glomerulonephritis in mice. Nephrol Dial Transplant 19: 1875- 1880.

[0193] Lentes, K.U. et al. (1999). Genomic organization and mutational analysis of the human UCP2 gene, a prime candidate gene for human obesity. J Recept Signal Transduct Res 19:229-244. [0194] Li, P. et al. (2005). Blocking of monocyte chemoattractant protein- 1 during tubulointerstitial nephritis resulted in delayed neutrophil clearance. Am J Pathol 167:637-649.

[0195] Luo, Y. and Prestwich, G.D. (2001). Hyaluronic acid-N-hydroxysuccinimide: a useful intermediate for bioconjugation. Bioconjug Chem 12: 1085-1088.

[0196] Madesh, M. (2005). Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis. J Cell Biol 170: 1079-1090.

[0197] Madsen, K.L. et al. (1998). Stanniocalcin: a novel protein regulating calcium and phosphate transport across mammalian intestine. Am J Physiol Gastrointest Liver Physiol 274:G96-G102.

[0198] Mandal, S. et al. (2005). Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila. Dev Cell 9:843-854.

[0199] Manome, Y. et al. (2000). Ultrasound facilitates transduction of naked plasmid DNA into colon carcinoma cells in vitro and in vivo. Hum Gene Ther 11: 1521-1528.

[0200] Maritim, A.C. et al. (2003). Diabetes, oxidative stress, and antioxidants: a review. J Biochem MoI Toxicol 17:24-38.

[0201] McCudden, CR. et al. (2002). Characterization of mammalian stanniocalcin receptors. Mitochondrial targeting of ligand and receptor for regulation of cellular metabolism. J Biol Chem 277:45249^5258.

[0202] McCudden, CR. et al. (1993). Co-localization of stanniocalcin- 1 ligand and receptor in human breast carcinomas. MoI Cell Endocrinol 213: 167-172, 2004Naito M: Macrophage heterogeneity in development and differentiation. Arch Histol Cytol 56:331-351.

[0203] Miller, A.D. (1990). Retrovirus packaging cells. Human Gene Therapy 1 :5-14.

[0204] Nishio, K. et al. (2005). Characterization of the differential expression of uncoupling protein 2 and ROS production in differentiated mouse macrophage-cells (MmI) and the progenitor cells (Ml). J MoI Histol 36:35-44.

[0205] Odobasic, D. et al. (2005). Glomerular expression of CD80 and CD86 is required for leukocyte accumulation and injury in crescentic glomerulonephritis. J Am Soc Nephrol 16:2012- 2022.

[0206] Olsen, H.S. et al. (1996). Human stanniocalcin: a possible hormonal regulator of mineral metabolism. Proc Natl Acad Sci USA 93: 1792-1796.

[0207] Paciga, M. et al. (2003). Targeting of big stanniocalcin and its receptor to lipid storage droplets of ovarian steroidogenic cells. J Biol Chem 278:49549^9554.

[0208] Pillai, O. and Panchagnula, R. (2001). Polymers in drug delivery. Curr Opin Chem Biol 5:447-451.

[0209] Roberts, MJ. et al. (2002). Chemistry for peptide and protein PEGylation." Adv Drug Deliv Rev 54:459-476.

[0210] Rodriguez-Iturbe, B. et al. (1981). Studies on the renin-aldosterone system in the acute nephritic syndrome. Kidney Int 19:445-453.

[0211] Rousset, S. (2004). The Biology of Mitochondrial Uncoupling Proteins. Diabetes 53:S130-S135. [0212] Sack, M.N. (2006). Mitochondrial depolarization and the role of uncoupling proteins in ischemia tolerance. Cardiovasc Res 72:210-219.

[0213] Sato, N. et al. (1998). Changes of gene expression by lysophosphatidylcholine in vascular endothelial cells: 12 up-regulated distinct genes including 5 cell growth-related, 3 thrombosis-related, and 4 others. JBiochem (Tokyo) 123: 1119-1126.

[0214] Schultz, B. E. (2001). Structures and proton-pumping strategies of mitochondrial respiratory enzymes. Annu Rev Biophys Biomol Struct 30:23-65.

[0215] Serlachius, M. et al. (2002). Expression of stanniocalcin-1 in megakaryocytes and platelets. Br J Haematol 119:359-363.

[0216] Sherratt, H.S. et al. (1988). Methods for study of normal and abnormal skeletal muscle mitochondria. Methods Biochem Anal 33:243-335.

[0217] Sheerin, N. S. et al. (2001). Protection and injury: the differing roles of complement in the development of glomerular injury. Eur J Immunol 31 : 1255-1260.

[0218] Sheerin, N.S. et al. (1997). TNF-alpha regulation of C3 gene expression and protein biosynthesis in rat glomerular endothelial cells. Kidney Int 51 : 703-710.

[0219] Sheikh-Hamad, D. et al. (2003). Stanniocalcin-1 is a naturally occurring L-channel inhibitor in cardiomyocytes: relevance to human heart failure. Am J Physiol Heart Circ Physiol 285:H442-H448.

[0220] Singh, N. et al. (1995). Oxidative stress and heart failure. MoI Cell Biochem \M\11- 81.

[0221] Sottocasa, G.L. et al. (1967). An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J Cell Biol 32:415- 438.

[0222] Stannius, H. (1939). Uber Nebenniere bei Knochenfischen. Arch Anat Physiol 6:91 - 101.

[0223] Tacke, F. et al. (2006). Immature monocytes acquire antigens from other cells in the bone marrow and present them to T cells after maturing in the periphery. J Exp Med 203:583- 597.

[0224] Tacke, F. et al. (2006). Migratory fate and differentiation of blood monocyte subsets. Immunobiology 211 :609-618.

[0225] Tagaki, Y. et al. (1985). Effects of the removal of the corpuscles of Stannius on the transport of calcium across the intestine of rainbow trout. Zool Sci 2:523-530.

[0226] Takahashi, K. et al. (1996). Development and heterogeneity of macrophages and their related cells through their differentiation pathways. Pathol Int 46:473-485.

[0227] Tergaonkar, V. (2006). NFkappaB pathway: a good signaling paradigm and therapeutic target. Int JBiochem Cell Biol 38: 1647-1653.

[0228] Thompson, M.P. et al. (2004). Links between fatty acids and expression of UCP2 and UCP3 mRNAs. FEBS Lett 568:4-9.

[0229] Tipping, P. G. et al. (1985). T lymphocyte participation in antibody-induced experimental glomerulonephritis. Kidney Int 27:530-537. [0230] Varghese, R. et al. (1998). Comparative analysis of mammalian stanniocalcin genes. Endocrinology 139:4714-4725.

[0231] Varghese, R. et al. (2002). Overexpression of human stanniocalcin affects growth and reproduction in transgenic mice. Endocrinology 143:868-876.

[0232] Veronese, F.M. and Harris, J.M. (2002). Introduction and overview of peptide and protein pegylation. Adv Drug Deliv Rev 54:453-456.

[0233] Vu, T.H. et al. (1998). Clinical manifestations of mitochondrial DNA depletion. Neurology 50: 1783-1790, 1998

[0234] Wada, Y. et al. (2000). Biological nano motor, ATP synthase F(o)F(l): from catalysis to gammaepsilonc(10-12) subunit assembly rotation. Biochim Biophys Acta 1459:499-505.

[0235] Wagner, G.F. et al. (1997). Development of a dose-response bioassay for stanniocalcin in fish. MoI Cell Endocrinol 128: 19-28.

[0236] Wang, Y. et al. (2002). Vitamin D(3)-up-regulated protein- 1 is a stress-responsive gene that regulates cardiomyocyte viability through interaction with thioredoxin. J Biol Chem 277:26496-26500.

[0237] Wang, Y. et al. (2006). Stanniocalcin- 1 regulates energy metabolism and superoxide production in macrophages. J Am Soc Nephrol 17:7 IA (SA-FC004).

[0238] Wendelaar Bonga, S.E. and Pang, P.K. (1991). Control of calcium regulating hormones in the vertebrates. Int Rev Cytol 128: 139-213.

[0239] Wendelaar Bonga, S.E. et al. (1989). Immunocytochemical localization of hypocalcin in the endocrine cells of the corpuscles of Stannius in three teleost species (trout, flounder and goldfish). Cell Tissue Res 255:651-656.

[0240] Wu, J. et al. (2002). CD4(+) T cells specific to a glomerular basement membrane antigen mediate glomerulonephritis. J Clin Invest 109:517-524.

[0241] Yang, N. et al. (1998). Reversal of established rat crescentic glomerulonephritis by blockade of macrophage migration inhibitory factor (MIF): potential role of MIF in regulating glucocorticoid production. MoI Med 4:413-424.

[0242] Yorek, M.A. (2003). The role of oxidative stress in diabetic vascular and neural disease. Free Radical Research 37:471-480.

[0243] Yoshiko, Y. et al. (2002). Stanniocalcin 1 (STCl) protein and mRNA are developmentally regulated during embryonic mouse osteogenesis: the potential of stcl as an autocrine/paracrine factor for osteoblast development and bone formation. J Histochem Cytochem 50:483^92.

[0244] Yu, X.Q. et al. (1999). IL-I up-regulates osteopontin expression in experimental crescentic glomerulonephritis in the rat. Am J Pathol 154:833-841.

[0245] Zhang, J. et al. (1998). Expression, purification, and bioassay of human stanniocalcin from baculovirus-infected insect cells and recombinant CHO cells. Protein Expr Purif 12:390- 398.

Claims

WHAT IS CLAIMED IS:

1. A method of protecting the heart in a mammal comprising administering a therapeutically effective amount of an active agent selected from the group consisting of stanniocalcin-1, a stanniocalcin- 1 derivative and a nucleic acid encoding stanniocalcin- 1 or stanniocalcin- 1 derivative to a mammal in need thereof to protect the heart.

2. The method of claim 1, wherein the active agent is administered to a mammal having hypertension in an amount to reduce inflammation and superoxide generation.

3. The method of claim 1, wherein the active agent is administered to a mammal suffering from a myocardial infarction in an amount to reduce inflammation and superoxide generation.

4. The method of claim 1 , wherein the active agent is administered to a mammal having inflammatory artery disease in an amount to reduce angiotensinogen-II-mediated superoxide generation.

5. The method of any one of claims 1 to 4, wherein the heart tissue is contacted with the active agent.

6. The method of any one of claims 1 to 4, wherein the active agent is administered topically, transdermally, subcutaneously, intra-nasally, intraveneously, parenterally, systemically or by tablet.

7. The method of claim 1, wherein the amount of stanniocalcin-1 or stanniocalcin-1 derivative that is administered is from about 10 ng/ml (10 ng/g) to about 500 ng/ml (500 ng/g)-

8. A method of reducing inflammation in a mammal comprising administering a therapeutically effective amount of an active agent selected from the group consisting of stanniocalcin-1, a stanniocalcin-1 derivative and a nucleic acid encoding stanniocalcin-1 or stanniocalcin-1 derivative to a mammal in need thereof to reduce inflammation.

9. The method of claim 8, wherein inflamed tissue or inflamed area of the mammal is contacted with the active agent.

10. The method of claim 8, wherein the active agent is administered topically, transdermally, subcutaneously, intra-nasally, intraveneously, parenterally, systemically or by tablet.

11. The method of claim 9, wherein the active agent is administered topically or transdermally.

12. The method of claim 8, wherein the amount of stanniocalcin-1 or stanniocalcin-1 derivative that is administered is from about 10 ng/ml (10 ng/g) to about 500 ng/ml (500 ng/g).

13. A method of reducing reactive oxidants in a mammal comprising administering a therapeutically effective amount of an active agent selected from the group consisting of stanniocalcin-1, a stanniocalcin-1 derivative and a nucleic acid encoding stanniocalcin-1 or stanniocalcin-1 derivative to a mammal in need thereof to reduce reactive oxidants.

14. The method of claim 13, wherein the active agent is administered topically, transdermally, subcutaneously, intra-nasally, intraveneously, parenterally, systemically or by tablet.

15. The method of claim 13, wherein the amount of stanniocalcin-1 or stanniocalcin-1 derivative that is administered is from about 10 ng/ml (10 ng/g) to about 500 ng/ml (500 ng/g).

16. A method of inducing expression of mitochondrial uncoupling proteins in cells of a mammal comprising administering a therapeutically effective amount of an active agent selected from the group consisting of stanniocalcin-1, a stanniocalcin-1 derivative and a nucleic acid encoding stanniocalcin-1 or stanniocalcin-1 derivative to a mammal in need thereof to induce the expression of mitochondrial uncoupling proteins.

17. The method of claim 16, wherein the cells are macrophages or heart cells.

18. The method of claim 17, wherein the active agent is administered topically, transdermal Iy, subcutaneously, intra-nasally, intraveneously, parenterally, systemically or by tablet.

19. The method of claim 17, wherein the amount of stanniocalcin-1 or stanniocalcin-1 derivative that is administered is from about 10 ng/ml (10 ng/g) to about 500 ng/ml (500 ng/g).

20. A method of promoting weight loss in a mammal comprising administering a therapeutically effective amount of an active agent selected from the group consisting of stanniocalcin-1, a stanniocalcin-1 derivative and a nucleic acid encoding stanniocalcin-1 or stanniocalcin-1 derivative to a mammal in need thereof to promote weight loss.

21. The method of claim 20, wherein the active agent is administered topically, subcutaneously, intra-nasally, systemically or by tablet.

22. The method of claim 20, wherein the amount of stanniocalcin-1 or stanniocalcin-1 derivative that is administered is from about 10 ng/ml (10 ng/g) to about 500 ng/ml (500 ng/g)"

23. The method of claim 20, wherein the amount of active agent decrease efficiency of food utilization.

24. A pharmaceutical composition comprising a heart protective effective amount of stanniocalcin-1, a stanniocalcin-1 derivative or nucleic acid encoding stanniocalcin-1 or a stanniocalcin-1 derivative and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24, wherein the composition is in the form of a subcutaneous depot.

26. A pharmaceutical composition comprising an anti-inflammatory effective amount of stanniocalcin-1, a stanniocalcin-1 derivative or nucleic acid encoding stanniocalcin-1 or a stanniocalcin-1 derivative and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26, wherein the composition is in the form of a subcutaneous depot.

28. A pharmaceutical composition comprising an anti-reactive oxidants effective amount of stanniocalcin-1, a stanniocalcin-1 derivative or nucleic acid encoding stanniocalcin-1 or a stanniocalcin-1 derivative and a pharmaceutically acceptable carrier.

29. The pharmaceutical composition of claim 28, wherein the composition is in the form of a subcutaneous depot.

30. A pharmaceutical composition comprising an effective amount of stanniocalcin-1, a stanniocalcin-1 derivative or nucleic acid encoding stanniocalcin-1 or a stanniocalcin-1 derivative and a pharmaceutically acceptable carrier, wherein the amount is effective to induce expression of mitochondrial uncoupling proteins.

31. The pharmaceutical composition of claim 30, wherein the composition is in the form of a subcutaneous depot.

32. A pharmaceutical composition comprising a weight loss promoting effective amount of stanniocalcin-1, a stanniocalcin-1 derivative or nucleic acid encoding stanniocalcin-1 or a stanniocalcin-1 derivative and a pharmaceutically acceptable carrier.

33. The pharmaceutical composition of claim 32, wherein the composition is in the form of a subcutaneous depot.