Use of KL1 to treat cardiovascular disease

Abstract

Compositions comprising peptides and dimers thereof that exhibit anti-fibrotic activity and decrease cardiac fibrosis in patients with Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD) are provided. In one embodiment the peptide is the KL1 domain of Klotho.

Description

USE OF KL1 TO TREAT CARDIOVASCULAR DISEASEFEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with government support under K23 DK115683-01 awarded by National Institutes of Health. The Government has certain rights in the invention.CRO S S-REFERENCE

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 710,384 filed October 22, 2024, which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0003] The application contains a Sequence Listing which has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy, created on October 21, 2025, is named IU-2025-032-02-WO_st26SL and is 5.56KB in size.BACKGROUND

[0004] Cardiac (or myocardial) fibrosis is a major contributor to cardiovascular-related morbidity and mortality in patients with advanced Chronic Kidney Disease (CKD). In postmortem examination, more than 90% of patients that had advanced CKD exhibited diffuse cardiac fibrosis which worsened with increasing hemodialysis vintage. Implications of myocardial fibrosis includes reduced left ventricular (LV) compliance, impaired LV diastolic filling, and development of diastolic heart failure. Furthermore, atrial and ventricular fibrosis substantially increases risk of arrythmias and sudden cardiac death in patients with CKD. CKD-associated factors, such as uremic toxins, pro-inflammatory cytokines, mineral disturbances, volume overload, and hypertension, can promote myocardial fibroblasts to undergo phenotypic differentiation into secretory myofibroblasts, in vivo. These transformed myofibroblasts excessively deposit extracellular matrix (ECM) components such as collagen in the interstitium and perivasculature. Applicant has previously showed that trimeric collagen was upregulated in hearts of donors with CKD, which was replicated in cardiac fibroblasts treated with mineral stressors. To-date, there is still an unmet need for medical therapies that can directly target cardiac fibrosis in the CKD population.

[0005] a-Klotho (hereafter, Klotho) is a powerful anti-aging protein that has been reported to confer cardioprotective effects. In humans, circulating Klotho levels decrease early beginning at CKD stage II, and progressively decline with lower glomerular filtration rate (GFR). Deficiency of total circulating Klotho in advanced CKD has been associated to cardiac fibrosis, and increased risk for adverse cardiovascular events, and cardiovascular and all-cause mortality. In animal studies, Klotho-deficient mice develop a phenotype similar to patients with advanced CKD, including extensive cardiac fibrosis and shortened lifespan. Conversely, overexpression of Klotho or administration of exogenous Klotho rescues the Klotho-deficient phenotype including ameliorating myocardial fibrogenesis and increasing lifespan in CKD. These striking biological discoveries provide strong rationale to investigate Klotho’ s therapeutic role in the treatment of cardiac fibrosis in CKD. Translating Klotho into a potential therapeutic, however, is mired by its large size (130kDa), its potential instability and highly pleiotropic nature that runs the risk of off-target effects. Klotho is formed by two domains, KL1 and KL2 (Fig. 1) and can exert both Fibroblast Growth Factor (FGF)23 -dependent and -independent effects. The former involves binding of the KL2 domain to FGF23, and this signaling has recently been shown to decrease bone volume and mineralization. Identification of the fragment of Klotho that could exert FGF23 -independent cardioprotective effects is therefore of critical importance for therapeutic targeting. Significantly, the KL1 domain of Klotho is not involved in FGF23 binding and therefore could be responsible for mediating FGF23 independent effects. Unfortunately, published cardiovascular experimental studies involving Klotho have been limited to examining its full-length form, therefore the isoform-specific effects of Klotho remain largely unknown. In fact, to-date no studies have investigated the functional role of KL1 in the cardiovascular system.

[0006] In accordance with one aspect of the present invention, a fragment of Klotho (SEQ ID NO: 2) is utilized to exert direct cardiovascular protective effects in CKD. Given that full-length Klotho has been shown to regulate the contractile phenotype in vascular smooth muscle cells, Applicant anticipates that the cardiovascular protective effects of KL1 involved a similar mechanism. This includes halting phenotype transformation and preservation of contractile myocardial fibroblasts into secretory myofibroblasts at the heart.SUMMARY

[0007] As disclosed herein, using a slowly progressive rat model of CKD, Applicant has discovered that 1) KL1 treatment significantly reduces total fibrosis in left ventricular hearts in CKD rats in vivo,' 2) Anti-fibrotic effects of KL1 were independent of alterations in renal function, mineral metabolism (including FGF23 and PTH levels), or collagen metabolism, in vivo, 3) KL1 directly inhibits myocardial cell fibrosis, in vitro,' and that 4) KL1 directly exerted anti-fibrotic effects by inhibiting myofibroblast transformation and preserving contractile cell phenotype, in vivo and in vitro,' 5) KL1 directly inhibits calcification of human aortic smooth muscle cells (HA-SMCs), in vitro.

[0008] In accordance with one embodiment, the present disclosure is directed to compositions and methods for reducing cardiac fibrosis and vascular smooth muscle cell calcification, and inhibiting myofibroblast transformation and preserving contractile cell phenotype, in a patient in need thereof. In one embodiment the patient administered the KL1 peptide is a patient with compromised kidney function, including patients with Chronic Kidney Disease (CKD). In one embodiment the method comprising administering to the patient a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a KL1 peptide, wherein the KL1 peptide is administered in an amount effective to decrease one or more of collagen type I, TGF-P, and a-SMA levels in cardiac fibroblast cells, without any changes in concentration of collagen type III, collagenases MMP1 and MMP9, or collagen cross-linking enzyme LOX in the patient’s fibroblast cells. In one embodiment the KL1 peptide comprises a truncated soluble Klotho protein (sFL-K) selected from the group consisting of SEQ ID NO: 2, a peptide having at least 95% sequence identity with SEQ ID NO: 2, or a peptide that differs from SEQ ID NO: 2, by one, two, or three amino acid substitutions. In a further embodiment the KL1 peptide can be modified to enhance the stability or retention times in a patient. In accordance with one embodiment the KL1 peptide is pegylated and / or acylated. In one embodiment the KL1 peptide is acylated with an acyl group comprising a C16-C18 fatty acid or C16-C18 fatty diacid chain.

[0009] In accordance with one embodiment the KL1 peptide is formulated with a pharmaceutically acceptable carrier for administration to the patient using any of the standard administrative routes. In one embodiment a pharmaceutical composition is prepared comprising a KL1 peptide of the present invention and a pharmaceutically acceptable carrier, wherein the composition is formulated for parenteral administration, optionally wherein the parenteral administration is by subcutaneous or intravenous administration.

[0010] In one embodiment, a method of providing a cardioprotective effects in a patient is provided, particularly a patient that has been diagnosed with Chronic Kidney Disease (CKD). The method comprises identifying a subject having kidney damage and / or decreased kidney function and administering to such an identified subject a KL1 peptide of the present invention. In accordance with one embodiment an improved method for treating myocardial fibrosis or other fibrotic diseases is provided. The method comprises the steps of administering to a patient a KL1 peptide as disclosed herein in an amount therapeutically effective for treating fibrotic diseases, including myocardial fibrosis.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0011] Fig. 1 Schematic of a-Klotho isoforms and Cy / + rat animal protocol and cohort, a-Klotho (or Klotho), can exist as membrane or circulating isoforms, in which the latter is further subdivided into cleaved soluble and alternatively spliced secreted isoforms. Soluble isoforms are produced from proteolytic cleavage of membrane full-length Klotho (mFL-K; SEQ ID NO: 1; MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWARFSRPPAPEAAGLFQGT FPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQ PATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRL LERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVK YWITIDNPYVVAWHGYATGRLAPGIRGSPRLGYLVAHNLLLAHAKVWHLYNTSFRPTQ GGQVSIALS SHWINPRRMTDHSIKECQKSLDF VLGWF AKPVFIDGDYPESMKNNLS SILP DFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLESPNLRQLLSWIDLEFNHPQIFI VENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRG YSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVVD NYIQVDTTLSQFTDLNVYLWDVHHSKRLIKVDGVVTKKRKSYCVDFAAIQPQIALLQE MHVTHFRFSLDWALILPLGNQSQVNHTILQYYRCMASELVRVNITPVVALWQPMAPNQ GLPRLLARQGAWENPYTALAFAEYARLCFQELGHHVKLWITMNEPYTRNMTYSAGHN LLKAHALAWHVYNEKFRHAQNGKISIALQADWIEPACPFSQKDKEVAERVLEFDIGWL AEPIFGSGDYPWVMRDWLNQRNNFLLPYFTEDEKKLIQGTFDFLALSHYTTILVDSEKE DPIKYNDYLEVQEMTDITWLNSPSQVAVVPWGLRKVLNWLKFKYGDLPMYIISNGIDD GLHAEDDQLRVYYMQNYINEALKAHILDGINLCGYFAYSFNDRTAPRFGLYRYAADQFEPKASMKHYRKIIDSNGFPGPETLERFCPEEFTVCTECSFFHTRKSLLAFIAFLFFASIISLS LIFYYSKKGRRSYK) at the al cut site to produce soluble full-length Klotho (sFL-K) or at both the al and a2 / cut sites to produce soluble KL1 (sKLl; SEQ ID NO: 2;EPGDGAQTWARFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIW DTFTHHPL APPGD SRNASLPLGAP SPLQP ATGD VASD S YNNVFRDTEALRELGVTHYRF SISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGG WANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGSPRL GYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDF VLGWFAKPVFIDGDYPESMKNNLSSILPDFTESEKKFIKGTADFFALCFGPTLSFQLLDPH MKFRQLESPNLRQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIMETL KAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLI EKNGFPPLPENQPL) and soluble (sKL2) isoforms. The secreted Klotho or secreted KL1 isoform (secKLl; SEQ ID NO: 3;MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWARFSRPPAPEAAGLFQGT FPDGFLW A VGSAAYQTEGGWQQHGKGASIWDTFTHHPL APPGD SRNASLPLGAP SPLQ PATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRL LERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVK YWITIDNPYVVAWHGYATGRLAPGIRGSPRLGYLVAHNLLLAHAKVWHLYNTSFRPTQ GGQVSIALS SHWINPRRMTDHSIKECQKSLDF VLGWF AKPVFIDGDYPESMKNNLS SILP DFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLESPNLRQLLSWIDLEFNHPQIFI VENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRG YSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVVD NYIQVSQLTKPISSLTKPYH) are produced from alternative (ALT) splicing of the mFL-K transcript. Amino acid numbers and sequences are based on the human protein sequence of Klotho. Majority of studies have focused on overexpressing or administering Klotho isoforms with an intact extracellular domain, or linked KL1-KL2 domains, which include the transmembrane (mFL-K) and soluble full-length Klotho (sFL-K) isoforms. Several studies have shown that Klotho isoforms containing the KL1 domain alone, such as the soluble KL1 (sKLl) and secreted KL1 (secKLl), are biologically active and exerts protective effects on various tissues. There is a paucity of studies investigating the effects of the lone KL2 domain such as in soluble KL2 (sKL2) isoform.

[0012] Figs. 2A and 2B. Cy / + rat animal protocol and cohort. Fig. 2A provides a timeline of casein diet, interventional treatment with intraperitoneal (IP) injections of either KL1 or vehicle, and euthanization in respect to rat age in weeks. Male Cy / + (CKD) rats were fed a casein diet at 22 weeks to produce a consistent CKD-MBD phenotype. At 27 weeks (correlating to stage 3B CKD in humans), treated with intraperitoneal injections of KL1 or vehicle solution. Fig. 2B provides a flow chart of initial and final numbers of rats in the study cohort. Rats that spontaneously expired or euthanized early due to declined rapidly in health with significant weight loss and moribund behavior prior to the 32 to 34-week endpoint were excluded from the analyses.

[0013] Figs.3A-3D. KLl-treated CKD rats exhibited reduced cardiac fibrosis, in vivo. Fig 3A provides histological imaging of left ventricular (LV) sections of rat hearts. Representative images of Masson’s Tri chrome staining of LV sections in rows 1 (mosaic of 5X images) and 2 (20X magnification) and H&E staining in rows 3 (mosaic of 5X images) and 4 (20X magnification). Black boxes indicate location of the 20X images within the LV tissue section. CKD rats developed significant interstitial and perivascular fibrosis which was reduced in KLl-treated rats. Fig.3B is a graph providing data on the quantified total area of fibrosis, or blue staining within Masson’s Trichrome stains, presented as percent area of whole LV tissue histological section (N=5 each group; one-way ANOVA and unpaired / -test applied). Fig.3C is a graph providing data on the left ventricular mass index (LVMI; WT 7V=11, CKD 7V=15, KL1 7V=17; Kruskal-Wallis and Kolmogorov-Smirnov tests applied) and Fig.3D is a graph providing data on the body weight (WT A=11, CKD A=15, KL1 JV=17; one-way ANOVA and unpaired t-test applied) measured at tissue harvesting. LVMI was determined by total heart weight divided by body weight. There was no significant difference in LVMI between KL1 and vehicle-treated CKD rats. CKD and KL1 rats had reduced body weight compared to WT rats. WT: healthy normal littermates, CKD: vehicle-treated (0.1% BSA in 0.9% saline) CKD rats, KL1: human recombinant KL 1 -treated (50pg / kg) CKD rats. ** <0.01, ***P<0.001, **** <0.0001, ns = nonsignificant. N = biological replicates. Scale bars: 1000pm x 1000pm (5X), 50pm x 50pm (20X).

[0014] Figs. 4A-4G. KLl-treated CKD rats had exhibited decreased pro-fibrotic cytokine, TGF-p, and myofibroblast marker, a-SMA, in vivo. Fig. 4A provides representative blot images for targets transforming growth factor-beta (TGF-[3, ~35 kDa). Semi-quantitativemeasurement of target protein immunoblot band intensities normalized to GAPDH. Fig. 4B is a graph demonstrating TGF-P expression was increased in CKD rats but decreased with KL1 treatment (WT =11, CKD #=13, KL1 #=12; one-way ANOVA and unpaired / -test applied).Fig. 4C provides representative blot images for targets alpha-smooth muscle actin (a-SMA, ~30 kDa), metalloproteinase 1 (MMP1, ~90 kDa), metalloproteinase 9 (MMP9, ~ 90kDa), and lysyl hydroxylase (LOX, ~70kDa) of rat left ventricular hearts. Semi-quantitative measurement of target protein immunoblot band intensities normalized to GAPDH. Fig. 4D is a graph demonstrating a-SMA was decreased in KL1 -treated rats vs. CKD and WT rats, but there was no significant difference in a-SMA between CKD and WT rats (WT #=11, CKD #=15, KL1 #=17; one-way ANOVA and unpaired / -test applied). There was no difference in expression of MMP1 (WT #=11, CKD #=15, KL1 #=17; one-way ANOVA and unpaired / -test applied), see Fig 4E, MMP9 (WT #=11, CKD #=15, KL1 #=17; one-way ANOVA and unpaired / -test applied), see Fig. 4F or LOX (WT #=11, CKD #=15, KL1 #=17, see Fig. 4G; Kruskal-Wallis and Kolmogorov-Smirnov tests applied) across all groups. *P<0.05, **P<0.01, ns = nonsignificant. N= biological replicates.

[0015] Figs. 5A-5E. Anti-fibrotic effects of KL1 did not involve alterations in renal function, in vivo. Interrogation of kidney function and structure included assessment of plasma blood urea nitrogen (See Fig. 5A: BUN; WT #=11, CKD #=15, KL1 #=17; average of n=2 per biological replicate; Kruskal -Wallis and Kolmogorov- Smirnov tests applied), serum creatinine (See Fig. 5B: WT #=11, CKD #=15, KL1 #=17; average of n=2 per biological replicate;Kruskal-Wallis and Kolmogorov-Smirnov tests applied), estimated glomerular fdtration rate (See Fig 5C: eGFR; WT #=11, CKD #=15, KL1 #=17; Kruskal-Wallis and Kolmogorov-Smirnov tests applied), and total weight of both kidneys relative to body weight (See Fig. 5D: WT #=11, CKD #=15, KL1 #=17; one-way ANOVA and unpaired / -test applied), eGFR was calculated by equations given in Besseling et al., which is dependent on creatinine, urea, and body weight. There was no statistical difference in normalized kidney weight, BUN, creatinine, or eGFR between KL1 -treated and CKD rats. Total fibrotic area was not significantly different between KLl-treated and CKD rats (See Fig. 5E: #=5 each group; one-way ANOVA and unpaired / -test applied). Quantification and histological imaging of mid-sections of rat kidneys was conducted.

[0016] Fig. 6A-6E KL1 treatment was not associated with any significant changes in mineral metabolism, in vivo. Fig. 6A: Plasma concentration of inorganic phosphorous (Pi) waselevated in CKD and KLl-treated rats compared to WT rats, but not significantly different between CKD and KLl-treated rats (WT V=11 , CKD V=15, KL1 =17; average of n=2 per biological replicate; one-way ANOVA and unpaired / -test applied). Fig. 6B: Plasma calcium was not significantly different across all groups (WT #=11, CKD #=15, KL1 #=17; average of n=2 per biological replicate; one-way ANOVA and unpaired / -test applied). Serum concentrations for Fig. 6C: intact and Fig.6D C-terminus fibroblast growth factor-23 (for both iFGF23 and cFGF23: WT #=11, CKD #=15, KL1 #=17; average of n=2 per biological replicate; Kruskal-Wallis and Kolmogorov- Smirnov tests applied); cFGF23 detects both intact and cleaved FGF23 and can be considered as a measurement of total FGF23. There was no significant difference in iFGF23 or cFGF23 between CKD and KLl-treated rats. Fig. 6E: Plasma iPTH was not statistically different between CKD and KL1 rats (WT #=11, CKD #=15, KL1 #=17; average of n=2 per biological replicate; Kruskal-Wallis and Kolmogorov-Smirnov tests applied). WT: healthy normal littermates, CKD: vehicle-treated (0.1% BSA in 0.9% saline) CKD rats, KL1: human recombinant KLl-treated (50pg / kg) CKD rats. ***7’<0.001, ****F><0.0001, ns = nonsignificant. #= biological replicates, n = technical replicates.

[0017] Figs. 7A-7F. KL1 treatment downregulated myofibroblast markers induced by mineral stressors, in vitro. Fig. 7A: Representative blot images and semi-quantitative measurement of immunoblot band intensity for targets Fig. 7B: collagen type I al chain (COL1A1, -200 kDa; #=2, n=6 per biological replicate; one-way ANOVA and unpaired / -test applied), Fig. 7C: collagen type III al chain (COL3A1, -150 kDa; N=2, n=6 per biological replicate; one-way ANOVA and unpaired / -test applied), Fig. 7D: COL1ALCOL3A1 ratio as an assessment of fibrosis (#=2, n=6 per biological replicate; one-way ANOVA and unpaired / -test applied), Fig. 7E: alpha-smooth muscle actin (a-SMA, -30 kDa; N=2, n=6 per biological replicate; one-way ANOVA and unpaired / -test applied), and Fig. 7F: metalloproteinase I (MMP1, -90 kDa; N=2, n=6 per biological replicate; one-way ANOVA and unpaired / -test applied) of human adult ventricular fibroblasts (HAVFs). Signal intensities of each protein target was normalized to GAPDH. Collagen type I and a-SMA were significantly reduced with KL1-treatment compared to vehicle treatment. G) Light microscopy images of HAVF cell culture before KL1 treatment (Day 0) and at end of treatment period (Day 5) revealed KL1 treatment helped preserve a spindle-like, non-secretory morphology.

[0018] Fig. 8. KL1 ameliorates calcification of human aortic smooth muscle cells, in vitro.Calcification was induced in human aortic smooth muscle cells (HASMCs) was induced with high phosphate and calcium (“mineral stressor”) media. KL1 decreased calcification in a dosedependent manner up to lOnM concentrations («=4-10 technical replicates per treatment per concentration group). One-way ANOVA and unpaired / -tests were applied. CTL, Control / Control Media; CM, Calcification Media. ****P<0.00Ql.

[0019] Fig. 9. Inflammatory marker levels in left ventricular rat hearts. Concentrations (pg / mL) and corresponding fluorescence signal intensity results of inflammatory markers from protein lysates of rat left ventricular heart sections. There was no difference in A) estimated protein concentrations or B) fluorescence signal intensities across all groups. Two-way ANOVA and multiple unpaired / -tests were applied.

[0020] Fig. 10. Full images of cropped representative immunoblots. Semi-quantification of target proteins was normalized to GAPDH loading control from the same blot. A) F4A, Figure 4A; F4C, Figure 4C; B) F7A, Figure 7A.DETAILED DESCRIPTION

[0021] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described herein, as such may vary. For the sake of brevity, the disclosures of the publications cited in this specification, including patents, are herein incorporated by reference. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in a patent, application, or other publication that is herein incorporated by reference, the definition set forth in this section prevails over the definition incorporated herein by reference.DEFINITIONS

[0022] In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.

[0023] The term "about" as used herein means greater or lesser than the value or range of values stated by 10 percent but is not intended to limit any value or range of values to only this broaderdefinition. Each value or range of values preceded by the term "about" is also intended to encompass the embodiment of the stated absolute value or range of values.

[0024] A “bioactive peptide” refers to peptides which can exert a biological effect in vitro and / or in vivo. As used herein a general reference to a peptide is intended to encompass peptides that have modified amino and carboxy termini. For example, an amino acid sequence designating the standard amino acids is intended to encompass standard amino acids at the N- and C- terminus as well as a corresponding hydroxyl acid at the N-terminus and / or a corresponding C-terminal amino acid modified to comprise an amide group in place of the terminal carboxylic acid.

[0025] As used herein an “acylated” amino acid is an amino acid comprising an acyl group which is non-native to a naturally occurring amino acid, regardless of the means by which it is produced. Exemplary methods of producing acylated amino acids and acylated peptides are known in the art and include acylating an amino acid before inclusion in the peptide or peptide synthesis followed by chemical acylation of the peptide. In some embodiments, the acyl group causes the peptide to have one or more of (i) a prolonged half-life in circulation, (ii) a delayed onset of action, (iii) an extended duration of action, and (iv) an improved resistance to proteases, such as DPP-IV.

[0026] As used herein, an “alkylated” amino acid is an amino acid comprising an alkyl group which is non-native to a naturally occurring amino acid, regardless of the means by which it is produced. Exemplary methods of producing alkylated amino acids and alkylated peptides are known in the art and including alkylating an amino acid before inclusion in the peptide or peptide synthesis followed by chemical alkylation of the peptide.

[0027] As used herein a "receptor" is a molecule that recognizes and binds with specific molecules in a high affinity interaction, producing some biological effect (either directly or indirectly) in a cell, or on the cells and / or tissues of the host organism. A "cellular receptor" is a molecule on or within a cell that recognizes and binds with specific molecules, producing some effect (either directly or indirectly) in the cell.

[0028] The term "identity" as used herein relates to the similarity between two or more sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100 to achieve a percentage. Thus, two copies of exactly the same sequence have 100% identity, whereas two sequences that have amino acid deletions, additions, or substitutions relative to one another have a lower degree of identity.Those skilled in the art will recognize that several computer programs, such as those that employ algorithms such as BLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J. Mol. Biol. 215:403-410) are available for determining sequence identity.

[0029] As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.

[0030] As used herein, the term "phosphate buffered saline" or "PBS" refers to aqueous solution comprising sodium chloride and sodium phosphate. Different formulations of PBS are known to those skilled in the art but for purposes of this invention the phrase "standard PBS" refers to a solution having have a final concentration of 137 mM NaCl, 10 mM Phosphate, 2.7 mM KC1, and a pH of 7.2-7.4.

[0031] As used herein the term "pharmaceutically acceptable salt" refers to salts of compounds that retain the biological activity of the parent compound, and which are not biologically or otherwise undesirable. Many of the compounds disclosed herein can form acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0032] As used herein, the term "treating" includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and / or preventing or eliminating said symptoms.

[0033] As used herein an "effective" amount or a "therapeutically effective amount" of a drug refers to a nontoxic but enough of the drug to provide the desired effect. The amount that is "effective" will vary from subject to subject or even within a subject overtime, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0034] The term, "parenteral" means not through the alimentary canal but by some other route such as subcutaneous, intramuscular, intraspinal, or intravenous.

[0035] As used herein an amino acid "substitution" refers to the replacement of one amino acid residue by a different amino acid residue.

[0036] As used herein, the term "conservative amino acid substitution" is defined herein as exchanges within one of the following five groups:I. Small aliphatic, nonpolar or slightly polar residues:Ala, Ser, Thr, Pro, Gly;II. Polar, negatively charged residues and their amides:Asp, Asn, Glu, Gin;III. Polar, positively charged residues:His, Arg, Lys; Ornithine (Orn)IV. Large, aliphatic, nonpolar residues:Met, Leu, He, Vai, Cys, Norleucine (Nle), homocysteine (hCys) V. Large, aromatic residues:Phe, Tyr, Trp, acetyl phenylalanine, napthylalanine (Nal)

[0037] As used herein the general term "polyethylene glycol chain" or "PEG chain", refers to mixtures of condensation polymers of ethylene oxide and water, in a branched or straight chain, represented by the general formula H(OCH2CH2)kOH, wherein k is at least 2.

[0038] As used herein the term "pegylated" and like terms refers to a compound that has been modified from its native state by linking a polyethylene glycol chain to the compound. A "pegylated polypeptide" is a polypeptide that has a PEG chain covalently bound to the polypeptide.

[0039] As used herein a "linker" is a bond, molecule or group of molecules that binds two separate entities to one another. Linkers may provide for optimal spacing of the two entities or may further supply a labile linkage that allows the two entities to be separated from each other. Labile linkages include photocleavable groups, acid-labile moieties, base-labile moieties, and enzyme-cleavable groups.

[0040] As used herein a "dimer" is a complex comprising two subunits covalently bound to one another via a linker. The term dimer, when used absent any qualifying language, encompasses both homodimers and heterodimers. A homodimer comprises two identical subunits, whereas a heterodimer comprises two subunits that differ.

[0041] As used herein the term "patient" without further designation is intended to encompass any warm blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs and other pets) and humans receiving a therapeutic treatment.

[0042] As used herein, the terms “including,” “containing,” and “comprising” are used in their open, non-limiting sense.

[0043] As used herein, the term “KL1 domain of Klotho” and “KL1 domain” defines the peptide of SEQ ID NO: 2, produced from proteolytic cleavage of membrane full-length Klotho (mFL-K; SEQ IDNO: 1;MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRAEPGDGAQTWARFSRPPAPEAAGLFQGT FPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQ PATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRL LERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVK YWITIDNPYVVAWHGYATGRLAPGIRGSPRLGYLVAHNLLLAHAKVWHLYNTSFRPTQ GGQVSIALS SHWINPRRMTDHSIKECQKSLDF VLGWF AKPVFIDGD YPESMKNNLS SILP DFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLESPNLRQLLSWIDLEFNHPQIFI VENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRG YSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVVD NYIQVDTTLSQFTDLNVYLWDVHHSKRLIKVDGVVTKKRKSYCVDFAAIQPQIALLQE MHVTHFRFSLDWALILPLGNQSQVNHTILQYYRCMASELVRVNITPVVALWQPMAPNQ GLPRLLARQGAWENPYTALAFAEYARLCFQELGHHVKLWITMNEPYTRNMTYSAGHN LLKAHALAWHVYNEKFRHAQNGKISIALQADWIEPACPFSQKDKEVAERVLEFDIGWL AEPIFGSGDYPWVMRDWLNQRNNFLLPYFTEDEKKLIQGTFDFLALSHYTTILVDSEKE DPIKYNDYLEVQEMTDITWLNSPSQVAVVPWGLRKVLNWLKFKYGDLPMYIISNGIDD GLHAEDDQLRVYYMQNYINEALKAHILDGINLCGYFAYSFNDRTAPRFGLYRYAADQF EPKASMKHYRKIIDSNGFPGPETLERFCPEEFTVCTECSFFHTRKSLLAFIAFLFFASIISLS LIFYYSKKGRRSYK) at the at both the al and a2 / p cut sites to produce soluble KL1, wherein the N-terminal signal sequence (MPASAPPRRPRPPPPSLSLLLVLLGLGGRRLRA) is maintained or removed (sKLl; SEQ ID NO: 2 presents the KL1 protein with the N-terminal signal sequence removed:EPGDGAQTWARFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIW DTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGG WANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGSPRL GYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDF VLGWFAKPVFIDGDYPESMKNNLSSILPDFTESEKKFIKGTADFFALCFGPTLSFQLLDPH MKFRQLESPNLRQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIMETL KAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLI EKNGFPPLPENQPL).

[0044] As used herein, the term “KL1 peptide” defines an N-terminal peptide fragment of soluble human a-Klotho (SEQ ID NO: 1), more particularly, comprising at least a 100 amino acid fragment of the first 472 amino acids of SEQ ID NO: 1, but does not include amino acid sequences of the KL2 domain. In one embodiment the KL1 peptide is a fragment of soluble human a-Klotho (SEQ ID NO: 1), comprising the sequence of SEQ ID NO: 2, a peptide having at least 90 or 95% sequence identity with SEQ ID NO: 2, or a bioactive fragment of a peptide having at least 90 or 95% sequence identity with SEQ ID NO: 2, but lacking amino acid sequences of the KL2 domain.

[0045] “Chronic Kidney Disease” (CKD) defines a condition in patients where kidney damage or reduced kidney function is detected, as indicated by an increased urine albumin-to-creatinine ratio or a decrease in glomerular filtration rate (eGFR) over an extended period of time (e.g. 3 months). For example, detecting a urine albumin > 30 mg / g creatinine or eGFR < 60 mL / min / 1.73 m2is diagnostic for CKD.EMBODIMENTS

[0046] In one embodiment a C-terminal truncated fragment of soluble Klotho (SEQ ID NO: 1) is provided that provides cardiovascular protective effects in patients in need thereof. The cardioprotective effects include reducing cardiac fibrosis and / or reducing arterial smooth muscle calcification in a patient. In one embodiment a C-terminal truncated fragment of soluble Klotho (SEQ ID NO: 1) is provided that provides a protective effects against cardiac fibrosis in patients suffering from Chronic Kidney Disease (CKD). In one embodiment the C-terminal truncated fragment of soluble Klotho comprises a 100, 200, 300 or 400 amino acid fragment of amino acids 1-472 of SEQ ID NO: 1, and excludes any amino acid sequence from the KL2 domain. In one embodiment the C-terminal truncated fragment of soluble Klotho comprises a peptide havingat least 85%, 90%, 95%, 99% sequence identity with SEQ ID NO: 2. In one embodiment the C-terminal truncated fragment of soluble Klotho comprises a peptide of SEQ ID NO: 2, or an amino acid sequence that differs from SEQ ID NO: 2 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions. Each of these peptide fragments of, or derivatives of, soluble Klotho, that exhibit the ability to decrease one or more of collagen type I, TGF-P, and a-SMA levels in cardiac fibroblast cells, are generally referenced as KL1 peptides. In one embodiment the KL1 peptide consists of SEQ ID NO: 2.

[0047] The present disclosure is the first to investigate the role of the peptide of SEQ ID NO: 2 on cardiac fibrosis in patients with CKD. As disclosed herein, the truncated KL1 domain of Klotho exhibits protective effects against cardiac fibrosis in CKD rats. Several studies have shown that full-length Klotho can ameliorate fibrosis and myofibroblast differentiation by inhibiting TGF-P signaling pathways in various tissues, including the heart, kidneys, and eyes. In a recent study that isolated a 30 amino acid-long peptide from the KL1 domain of Klotho, this fragment was shown to directly inhibit TGF-P signaling by competitively binding to TGF-P receptor 2 in rat kidney interstitial fibroblasts (Yuan et al., Nat Commun 2022;13:438 and Chen et al,. Kidney Int 2022;102:506-520). However, these peptides predominantly constitute the internal structure (or non-solvent exposed surface area) of the KL1 domain and are preferentially distributed to the diseased kidney instead of the heart, in vivo, and may not reflect similar cardioprotective mechanisms as the full KL1 domain. The data disclosed herein provides new insight and discovery that the KL1 domain harnesses anti-fibrotic effects at the heart, and these properties are independent of alterations in mineral or collagen metabolism, or changes in kidney function.

[0048] Full-length Klotho can inhibit cardiac hypertrophy by binding FGF23 with FGFRlc. Although the KL1 domain cannot interact with FGF23, the KL1 domain can bind lipid rafts to inhibit PI3K-Akt signaling leading to inhibition of cardiomyocyte hypertrophy. As disclosed herein Applicant found no difference in LVMI with the administration of the KL1 domain, which suggests the dosage of the KL1 domain to achieve anti-fibrotic effects at the heart is insufficient to inhibit PI3K-Akt signaling. Furthermore, Applicant did not detect any changes in elevated Pi levels, which can exacerbate cardiac hypertrophy and fibrosis. The lack of reduction in Pi is consistent with the expectation that the KL1 domain does not bind with FGF23 and FGFRlc to induce phosphaturic effects. Although the KL1 domain, like full length Klotho, mayexert P-glucuronidase activity to modify glycans on sodium-dependent phosphate transporters to induce phosphaturic effects, the results disclosed herein strengthen the notion that this may not be sufficient enough to result in a significant reduction in total phosphate. Elevated intracellular calcium and excess PTH can also contribute to cardiac fibroblast activation and fibrogenesis. Klotho regulates calcium handling at the cellular level by regulating epithelial calcium channels via its sialidase activity (e.g., downregulates transient receptor potential cation channel subfamily C member 6 (TRPC6) and retains cell surface presence subfamily V member 5 (TRPV5)). In the parathyroid gland, Klotho can indirectly affect blood calcium levels by suppressing PTH production via binding to calcium sensing receptors (CaSR) and FGF23 / FGFR1 signaling, or promote PTH production by binding to Na+ / K+-ATPase al subunit. However, studies that have overexpressed or knocked out Klotho showed no significant change in serum calcium levels. Consistent with these findings no changes in plasma calcium nor intact PTH were detected upon administration of the KL1 domain.

[0049] Cardiac fibrosis is characterized by increase in COL1 / 3 ratio, which is primarily driven by increased COL1 synthesis. Fibrotic donor hearts from patients with advanced CKD exhibited increase in COL1 and decrease in COL3 in recipient hosts. As disclosed herein, human adult ventricular fibroblasts (HAVFs) exposed to high mineral stress increased COL1 which was reduced by the KL1 domain, in vitro. This was accompanied by reduced a-SMA without changes in metalloproteinase I expression compared to vehicle control. This suggests that the KL1 domain does not impact collagen metabolism to reduce COL1 but may reduce COL1 via inhibiting the secretory myofibroblast phenotype in the context of CKD. However, these findings do not rule out that the KL1 domain may alter collagen metabolism in the context of other diseases and tissues. Takenaka et al. found that transcriptional expression of COL1 was reduced in the kidneys of PKD mice (Takenaka et al., Am J Physiol Renal Physiol 2020;318:F557-F564). Qiu et al. found that MMP1 and MMP3 were reduced in KL1 -treated pelvic floor fibroblasts (Qiu et al., Med Sci Monit 2019;25:3815-3824). It is possible that the uremic milieu of the Cy / + rat may mask some of KLl’s effects. Chronic low-grade inflammation is a common complication in CKD that can contribute to cardiac fibrosis, and previous studies have shown that the KL1 domain can suppress myocardial pro-inflammatory cytokines in endotoxemia animal models. Although the model presented herein suggests inflammation is not involved in anti-fibrotic effects of KL1, Applicant has discovered that the KL1 domain can reduce cardiacfibrosis through non-inflammatory pathways. Additionally, Applicant demonstrated that the KL1 domain reduced the secretory myofibroblast marker, a-SMA, in vivo, suggesting that the KL1 domain induces a phenotypic change in cardiac fibroblasts away from a secretory myofibroblast phenotype. TGF-P can promote phenotypic transformation of fibroblasts into myofibroblasts by upregulating a-SMA via focal adhesion kinase (FAK) and integrin-dependent pathways; a-SMA is critical to integrin-induced activation of TGF-0. By occupying TGF-P receptors and preventing a-SMA upregulation, the KL1 domain may inhibit further TGF-P activation and propagation of fibrosis in the heart. a-SMA also mechanically regulates TRPC6-mediated Ca2+ which modulates COL1 expression. Therefore, the KL1 domain may inhibit cardiac fibrosis as an upstream regulator of a-SMA.

[0050] By utilizing the Cy / + model, a well-established slowly progressive model that reflects human progressive CKD-MBD, Applicant has discovered that the KL1 domain is a biologically active component that harnesses cardioprotective effects of Klotho in CKD. Furthermore, Applicant demonstrates that the KL1 domain exhibits similar anti-fibrotic effects in a validated in vitro model of cardiac fibrosis in CKD. In both CKD models, the KL1 domain exerts anti-fibrotic effects via inhibition of the secretory myofibroblast phenotype. Furthermore, KLl’s effects may be limited by initiation at 27 weeks of age in the model system, which corresponds to approximately Stage 3B human CKD. Starting the treatment regimen at an earlier stage of CKD or a higher dosage of KL1 may result in a greater protective effect.

[0051] Applicant has demonstrated that the KL1 domain can inhibit cardiac fibrosis in rats with CKD-MBD, in vivo,' KLl’s anti-fibrotic effects occur at the phenotypic level of regulation as evidenced by reduced a-SMA rather than regulation of collagen metabolism, in vivo and in vitro, these protective effects are independent of FGF23, kidney function, and mineral regulation alterations, in vivo,' and the KL1 domain can inhibit myocardial cell fibrosis induced by mineral stressors found in CKD, in vitro.

[0052] In accordance with one embodiment of the present disclosure, a method for reducing cardiac fibrosis in a patient in need thereof is provided, wherein the method comprises administering to said patient a pharmaceutical composition comprising a KL1 peptide in an amount effective to decrease one or more of collagen type I, TGF-P, and a-SMA levels in cardiac fibroblast cells. Furthermore, in one embodiment, the KL1 peptide is administered in an amount effective to decrease each of collagen type I, TGF-P, and a-SMA levels in cardiacfibroblast cells without inducing any changes in collagen type III, collagenases MMP1 and MMP9, or collagen cross-linking enzyme LOX in cardiac fibroblast cells. In accordance with one embodiment a method for reducing cardiac fibrosis in a patient with Chronic Kidney Disease is provided wherein the method comprises administering to said patient a pharmaceutical composition comprising a KL1 peptide and a pharmaceutically acceptable carrier. In one embodiment the administered KL1 peptide is the KL1 domain.

[0053] In accordance with one embodiment the administered KL1 peptide comprises a C-terminal truncated fragment of soluble Klotho (SEQ ID NO: 1) that induces a protective effect against cardiac fibrosis in patients suffering from Chronic Kidney Disease (CKD). In one embodiment the C-terminal truncated fragment of soluble Klotho comprises a 100, 200, 300 or 400 amino acid fragment of amino acids 1-472 of SEQ ID NO: 1. In one embodiment the C-terminal truncated fragment of soluble Klotho comprises a peptide having at least 85%, 90%, 95%, 99% sequence identity with amino acids 1-472 of SEQ ID NO: 1. In one embodiment the KL1 peptide comprises a peptide of SEQ ID NO: 2, or an amino acid sequence that differs from SEQ ID NO: 2 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, wherein the KL1 peptide is devoid of any amino acid sequence of the KL2 domain. In one embodiment the KL1 peptide comprises an amino acid selected from the group consisting of SEQ ID NO: 2, or a peptide that differs from SEQ ID NO: 2, by one or two amino acid substitutions. In one embodiment the KL1 peptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 wherein the KL1 peptide is devoid of any amino acid sequence of the KL2 domain.

[0054] The KL1 peptides of the present disclosure can be further modified to have an improved therapeutic index and an in vivo extended time of action when administered to a warm blooded mammal including, for example, homo sapiens. More particularly, in one embodiment the peptides and dimers disclosed herein are modified by the covalent linkage of a fatty acid or fatty diacid of sufficient size to bind serum albumin with high affinity, optionally wherein the fatty acid or fatty diacid a C16-C18 fatty acid or C16-C18 fatty diacid. In accordance with one embodiment one or more lysine resides of the KL1 peptide or dimer disclosed herein is modified by the covalent linkage of a C16-C18 fatty acid or C16-C18 fatty diacid the side chain of the lysine. In one embodiment the acylated lysine residue is a lysine that has been added to the amino or carboxy terminus of a peptide or dimer of the present disclosure. In accordance with one embodiment the peptides and dimers disclosed herein are further modified by acylation,wherein the acyl group is linked to the side chain of an amino acid, optionally lysine or cysteine, located at the N-terminus and / or at the C-terminus of the peptide or dimer. In one embodiment the acyl group is of sufficient size to bind serum albumin with high affinity. In one embodiment the acyl group is a C16-C18 fatty acid or C16-C18 fatty diacid, optionally wherein the acyl group is linked via a spacer. In another embodiment the peptide is modified by pegylation of the peptide.

[0055] The compositions of the present disclosure can be administered using any of the standard routes of administration including oral, intranasal administration, intradermal administration, and parenteral administration. In particular, the parenteral administration can be a cutaneous, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. In one embodiment the peptides of the present disclosure are administered parenterally, optionally via intravenous injection.

[0056] In accordance with one embodiment a method of treating a patient with Chronic Kidney Disease (CKD) to reduce associated cardiac fibrosis is provided. In accordance with this method a patient who has damage to their kidneys and / or reduced kidney function is identified as a patient at risk for developing cardiac fibrosis, characterized by an increased collagen type I deposition as well as cardiac fibroblast activation and differentiation into myofibroblasts. Such identified patients can be treated by the administration of a KL1 peptide, including for example the KL1 domain, to reduce or prevent collagen type I deposition in cardiac muscle of the patient and decrease or prevent cardiac fibrosis relative to patients not receiving such treatment. In accordance with one embodiment a method of reducing cardiac fibrosis associated patient having Chronic Kidney Disease (CKD) comprises the steps of identifying a patient with CKD and administering to said identified patient a pharmaceutical composition comprising a KL1 peptide of the present disclosure, wherein said peptide decreases collagen type I, TGF-P, and / or a-SMA concentrations in human cardiac fibroblast cells upon contact of fibroblast cells with the KL1 peptide. In one embodiment the KL1 peptide decreases the concentration of each of collagen type I, TGF-P, and a-SMA in human cardiac fibroblast cells upon contact of fibroblast cells with the KL1 peptide. In one embodiment the KL1 peptide comprises an amino acid selected from the group consisting of SEQ ID NO: 2, or a peptide that differs from SEQ ID NO: 2, by one or two amino acid substitutions or a peptide having at least 95% sequence identity to SEQ ID NO: 2.

[0057] In accordance with one embodiment a pharmaceutical composition is provided comprising aKLl peptide and a pharmaceutically acceptable carrier, wherein the KL1 peptide inhibits or prevents cardiac fibrosis in a patient having CKD upon administration of the KL1 peptide to the patient. In one embodiment a pharmaceutical composition is provided comprising a KL1 peptide and a pharmaceutically acceptable carrier, wherein the administered KL1 peptide comprises a C-terminal truncated fragment of soluble Klotho (SEQ ID NO: 1) that induces a protective effect against cardiac fibrosis in patients suffering from Chronic Kidney Disease (CKD). In one embodiment the C-terminal truncated fragment of soluble Klotho comprises a 100, 200, 300 or 400 amino acid fragment of SEQ ID NO: 2. In one embodiment the C-terminal truncated fragment of soluble Klotho comprises a peptide having at least 85%, 90%, 95%, 99% sequence identity with SEQ ID NO: 2. In one embodiment the KL1 peptide comprises a peptide of SEQ ID NO: 2, or an amino acid sequence that differs from SEQ ID NO: 2 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions. In one embodiment the KL1 peptide comprises an amino acid selected from the group consisting of SEQ ID NO: 2, or a peptide that differs from SEQ ID NO: 2, by one or two amino acid substitutions. In one embodiment the KL1 peptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. In one embodiment a dimer is formed between two KL1 peptides. In one embodiment the pharmaceutical composition comprises a KL1 peptide that has been further modified by pegylation or acylation to enhance retention times and therapeutic index after administration to a patient. In one embodiment the pharmaceutical composition comprises a KL1 peptide that is acylated with an acyl group comprising a C16-C18 fatty acid or C16-C18 fatty diacid chain. In accordance with one embodiment the pharmaceutical compositions of the present disclosure are administer to a patient as a method preventing, or decreasing the rate of cardiac fibrosis in a patient having CKD.

[0058] In accordance with one embodiment a pharmaceutical composition is provided comprising any of the KL1 peptides or dimers disclosed herein, preferably at a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceutically acceptable diluent, carrier or excipient. In one embodiment the pharmaceutical compositions comprise aqueous solutions that are sterilized and optionally stored within various package containers. In other embodiments the pharmaceutical compositions comprise a lyophilized powder. The pharmaceutical compositions can be further packaged as part of a kit that includesa disposable device for administering the composition to a patient. The containers or kits may be labeled for storage at ambient room temperature or at refrigerated temperature.

[0059] The pharmaceutical compositions of the present disclosure may be administered by any convenient route, that suits the desired therapy. Preferred routes of administration include oral administration, in particular in tablet, in capsule, in dragee, in powder, or in liquid form, intranasal administration, intradermal administration, and parenteral administration, in particular cutaneous, subcutaneous, intramuscular, intraperitoneal, or intravenous injection. The pharmaceutical composition of the invention can be manufactured by the skilled person by use of standard methods and conventional techniques appropriate to the desired formulation. When desired, compositions adapted to give sustained release of the active ingredient may be employed.

[0060] In one embodiment the kit is provided with a device for administering the composition to a patient. The kit may further include a variety of containers, e.g., vials, tubes, bottles, and the like. Preferably, the kits will also include instructions for use. In accordance with some embodiments the device of the kit is an aerosol dispensing device, wherein the composition is prepackaged within the aerosol device. In another embodiment the kit comprises a syringe and a needle, and in some embodiments the KL1 peptide composition is prepackaged within the syringe.

[0061] Exemplary Embodiments

[0062] In accordance with embodiment 1, a method for reducing cardiac fibrosis and / or reducing arterial smooth muscle calcification in a patient in need thereof is provided, wherein said method comprises administering to said patient a pharmaceutical composition comprising a KL1 peptide in an amount effective to decrease one or more of collagen type I, TGF-0, and a-SMA levels in cardiac fibroblast cells, or an amount effective to reduce calcium levels in arterial smooth muscle cells, optionally wherein said decrease occurs without any changes in collagen type III, collagenases MMP1 and MMP9, or collagen cross-linking enzyme LOX.

[0063] In accordance with embodiment 2, the method of embodiment 1 is provided wherein the patient has Chronic Kidney Disease.

[0064] In accordance with embodiment 3, the method of embodiment 1 or 2 is provided wherein the KL1 peptide comprises at least a 100 amino acid fragment of SEQ ID NO: 2.

[0065] In accordance with embodiment 4, the method of any one of embodiments 1 to 3 is provided wherein the KL1 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, or a peptide that differs from SEQ ID NO: 2, by one or two amino acid substitutions.

[0066] In accordance with embodiment 5, the method of embodiment 1 or 2 is provided wherein the KL1 peptide comprises or consists of a 100, 200, 300 or 400 amino acid fragment of SEQ ID NO: 2, or comprises or consists of an amino acid sequence having at least 85%, 90%, 95%, 99% sequence identity with SEQ ID NO: 2, or comprises or consists of an amino acid sequence of SEQ ID NO: 2, or comprises an amino acid sequence that differs from SEQ ID NO: 2 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions.

[0067] In accordance with embodiment 6, the method of embodiment 1 or 2 is provided wherein the KL1 peptide comprises an amino acid having at least 95% sequence identity to SEQ ID NO: 2.

[0068] In accordance with embodiment 7, the method of any one of embodiments 1 to 6 is provided wherein the KL1 peptide is acylated with an acyl group, optionally wherein the acyl group comprises a C16-C18 fatty acid or C16-C18 fatty diacid chain.

[0069] In accordance with embodiment 8, the method of any one of embodiments 1 to 7 is provided wherein said peptide is administered parenterally, optionally via intravenous injection.

[0070] In accordance with embodiment 9, a method of treating a subject with impaired kidney function is provided, including treating subjects having Chronic Kidney Disease (CKD), to reduce associated cardiac fibrosis wherein the method comprises identifying a patient with impaired kidney function (e.g., CKD) and administering to said identified patient a pharmaceutical composition comprising a KL1 peptide of any one of embodiments 3-7, wherein said peptide decreases collagen type I, TGF-p, and / or a-SMA concentrations in human cardiac fibroblast cells upon contact of said fibroblast cells with said KL1 peptide.

[0071] In accordance with embodiment 10, the method of embodiment 9 is provided wherein the KL1 peptide comprises an amino acid selected from the group consisting of SEQ ID NO: 2, or a peptide that differs from SEQ ID NO: 2, by one or two amino acid substitutions.

[0072] In accordance with embodiment 11, the method of embodiment 9 is provided wherein the KL1 peptide comprises an amino acid having at least 95% sequence identity to SEQ ID NO: 2.

[0073] In accordance with embodiment 12, a method of inhibiting myofibroblast transformation and preserving contractile cell phenotype in a patient is provided wherein the method comprises identifying a patient with impaired kidney function (e.g., CKD) and administering to the identified patient a pharmaceutical composition comprising a KL1 peptide of any one of embodiments 3-7.

[0074] In accordance with embodiment 13, the method of embodiment 12 is provided wherein the KL1 peptide comprises an amino acid selected from the group consisting of SEQ ID NO: 2, or a peptide that differs from SEQ ID NO: 2, by one or two amino acid substitutions.

[0075] In accordance with embodiment 14, the method of embodiment 12 is provided wherein the KL1 peptide comprises an amino acid having at least 95% sequence identity to SEQ ID NO: 2.

[0076] In accordance with embodiment 15, a pharmaceutical composition comprising a KL1 peptide of any one of embodiments 3-7 and a pharmaceutically acceptable carrier is provided, wherein the peptide decreases collagen type I, TGF-P, and / or a-SMA concentrations in human cardiac fibroblast cells upon contact of said fibroblast cells with said KL1 peptide.

[0077] In accordance with embodiment 16, the composition of embodiment 15 is provided wherein the KL1 peptide comprises an amino acid selected from the group consisting of SEQ ID NO: 2, or a peptide that differs from SEQ ID NO: 2, by one or two amino acid substitutions.

[0078] In accordance with embodiment 17, the composition of embodiment 15 is provided wherein the KL1 peptide comprises an amino acid having at least 95% sequence identity to SEQ ID NO: 2.EXAMPLES EXAMPLE 1Effects of KL1 on cardiac fibrosis in CKD ratsAnimal Model

[0079] Male rats from the Cy / +IU colony established at the Indiana University (IU) School of Medicine were used in these experiments. Cy / + rats (hereafter, CKD rats) are of Hans:SPRD background with a spontaneous missense mutation in the Anks6 gene resulting in R823W substitution within the sterile alpha motif (SAM) domain of the SamCystin protein and formation of renal cysts. In turn, male CKD rats exhibit an autosomal dominant polycystic kidney disease(ADPKD) phenotype and spontaneously develop Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD) around 20 weeks-old and reach late-stage CKD-MBD with severe uremia by 33-34 weeks-old. This progressive CKD animal model recapitulates cardiovascular characteristics seen in patients with advanced CKD including myocardial fibrosis. The model results in mineral dysregulation that can contribute to adverse cardiac remodeling directly and indirectly by elevation of mineralotropic hormones, FGF23 and Parathyroid Hormone (PTH). Female Cy / + rats do not develop CKD-MBD despite an ovariectomy and were not used in this study.

[0080] Animal treatment protocol and the total rats included in the study are illustrated in Figs.2A and 2B, respectively. Briefly, all male rats (n=51) were switched from regular chow to a casein diet containing 18% casein protein, 0.7% inorganic phosphorous (Pi) and 0.6% calcium (Ca2+) (TD.04539, Envigo Teklad Diet) at 22 weeks-old to produce a more consistent CKD-MBD phenotype in CKD rats. By 27 weeks-old, CKD rats develop biochemical changes and decline to approximately 40% of normal renal function that correlate to CKD stage 3B in humans. At this age, treatment was administered daily via intraperitoneal injection with either 50pg / kg KL1 (“KL1” n=20; human recombinant Klotho, 100-53, PeproTech) or vehicle solution as placebo (“CKD” n=20; 0.1% bovine serum albumin (BSA) in 0.9% saline). KL1 was purchased from PeproTech and produced in Chinese Hamster Ovary (CHO) cell line. The 50 pg / kg dosage was used to match equimolar or greater effective concentrations of Klotho and KL1 in published studies. Wild-type littermates were used as healthy controls (“WT” n=l 1).

[0081] Rats were euthanized and harvested for tissue between 32-34 weeks-old (correlated to CKD stage 5) depending on the health condition of the rat. For euthanasia, rats were placed under deep plane of surgical anesthesia with 5% isoflurane; the thoracic cavity was then opened, and the full recoverable amount of circulating blood was collected from the inferior vena cava prior to intracardiac exsanguination and perfusion with 0.9% saline. Any rat that declined rapidly in health with moribund behavior and significant weight loss, or suddenly expired prior to reaching 32 to 34-week endpoint were excluded from the study. Left ventricular mass index (LVMI) was calculated based on total heart weight and rat body weight. Left ventricles were excised and separated in halves for further histological and protein analyses. All procedures were approved by the Indiana University School of Medicine Institutional Animal Care and UseCommittee and conform to the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.Blood Biochemistry

[0082] Rat plasma from a tail bleed prior to anesthesia for euthanasia was analyzed for blood urea nitrogen (BUN) (DIUR-100, BioAssay Systems), Ca2+ (C7503-480, Pointe Scientific), Pi (7516-500, Pointe Scientific), and intact parathyroid hormone (iPTH) (60-2500, Quidel). Rat serum collected during euthanasia was analyzed for creatinine (DICT-500, BioAssay Systems), intact (iFGF23) (60-6800, Quidel) and C-terminal (cFGF23) (60-6300, Quidel) fibroblast growth factor 23. Rat estimated glomerular filtration rate (eGFR) was calculated based on serum creatinine, plasma BUN, and body weight.Histology

[0083] Left ventricular sections of rat hearts were excised and fixed in 10% neutral buffered formalin solution (SF 100-4, Fisher Scientific) for at least 3 days and then transferred into 70% ethanol (64-17-5, Decon Labs) prior to H&E and Masson’s Tri chrome staining. Whole tissue images were created with Stitching Fiji / ImageJ plugin. Fibrosis severity represented by blue staining of Masson’s Trichrome was quantified with the Color Deconvolution ImageJ plugin method. Total tissue area was calculated via the Default Color Thresholding method tool in ImageJ.Inflammatory Marker Sandwich ELISA

[0084] Inflammatory markers from rat left ventricular hearts were measured using Quantibody® Rat Inflammation Array 1 Kit (QAR-INF-1-2, RayBiotech) as per manufacturer’s protocol. Upon completion of assay, assay slides were sent to manufacturer’s Array Scanning and Analysis service.Cardiac Fibroblast Cell Culture

[0085] Human adult ventricular fibroblasts (HAVFs) were sourced from 2 different adult donors (CC-2904, Lonza). HAVFs were cultured with FGMTM-3 Cardiac Fibroblast Growth Medium-3 BulletKitTM (CC-4526, Lonza) in a 5% CO2 and 37°C incubator. Applicant utilized an in vitromodel of CKD-related cardiac fibrosis previously established by our group. Briefly, HAVFs were pre-treated in high glucose DMEM (hgDMEM, 12100046, Gibco) with 2.5% FBS (10470328, Gibco) and 1.0% Penicillin / Streptomycin Solution (0503, ScienCell). For mineral stressor media, calcium chloride dihydrate (CaCh, C70-500, Fisher Scientific) and 0-glycerophosphate disodium salt pentahydrate (0-GP, 35675-50GM, EMD Millipore) were dissolved in Milli-Q ultrapure water and supplemented into hgDMEM to the final concentrations of 2.0mM CaCh and 3.8mM 0-GP. Alkaline phosphatase (ALP, Promega, Cat. M182A) was added to a final concentration of 0.25 U / mL. HAVFs were treated for 1 day with 5nM human recombinant KL1 (100-53, PeproTech) only and then for 5 days with both 5nM KL1 and mineral stressor media. KL1 was reconstituted to 0.1 pg / mL in vehicle solution (0.1% BSA in PBS). Cytologic evaluation was performed with Leica DMil microscope and Leica MCI 70 HD camera.Human Aortic Smooth Muscle Cell Culture

[0086] Human Aortic Smooth Muscle Cells (HASMCs) were cultured in a 5% CO2 and 37°C incubator. Applicant utilized an in vitro model of CKD-related vascular calcification previously established by our group. Briefly, HASMCs were pre-treated in high glucose DMEM (hgDMEM, 12100046, Gibco) with 5% FBS (10470328, Gibco) and 1.0% Penicillin / Streptomycin Solution (0503, ScienCell). For mineral stressor media, calcium chloride dihydrate (CaCh, C70-500, Fisher Scientific) and 0-glycerophosphate disodium salt pentahydrate (0-GP, 35675-50GM, EMD Millipore) were dissolved in Milli-Q ultrapure water and supplemented into hgDMEM to the final concentrations of 2.8mM CaCh and 3.8mM 0-GP. Alkaline phosphatase (ALP, Promega, Cat. M182A) was added to a final concentration of 1 U / mL HAVFs were treated for 1 day with human recombinant KL1 (100-53, PeproTech) only and then daily for 15 days with both KL1 and mineral stressor media. KL1 was reconstituted to 0.1 pg / mL in vehicle solution (10% glycerol in PBS).Antibodies

[0087] Primary antibodies used: COL1A1 (PA5-29569, Invitrogen), Collagen III [FH-7A] (MAI-22147, Invitrogen), TGF-0 (3711, Cell Signaling Technology), a-Smooth Muscle Actin (A5228, Sigma-Aldrich; 80008-1-RR, Proteintech), MMP1 (10371-2-AP, Proteintech), MMP9[EPl 254] (ab76003, Abeam), LOX (PAI-16955, Invitrogen), and GAPDH [14C10] (2118, Cell Signaling Technology). Secondary antibodies used: Anti-rabbit IgG (7074, Cell Signaling Technology) and Anti-mouse IgG (7076, Cell Signaling Technology). Detailed methods for immunoblotting are provided in the Supplementary Methods.Statistical Analysis

[0088] Data was analyzed and visualized with GraphPad Prism vlO.1.2 software. Results are expressed as either mean ± standard deviation or median [interquartile range]. Data was assessed for normality via Normal QQ plots and the Shapiro-Wilk tests due to N<50 total biological replicate sample size. If the data was not normally distributed, multi-group and pairwise comparisons were performed using Kruskal-Wallis and Kolmogorov-Smirnov tests, respectively. If the data was normally distributed, multi -group and pairwise comparisons were performed using one-way ANOVA or unpaired Student’s t-test, respectively. P-values <0.05 were considered statistically significant.RESULTS KL1 inhibits left ventricular fibrosis of CKD rats, in vivo

[0089] Out of a total of 51 male rats, 20 CKD rats were designated for KL1 treatment (“KL1”), 20 CKD rats for placebo (“CKD”), and 11 healthy littermates (“WT”) were used as controls (Fig.2B). Prior to initiation of the treatment period at 27 weeks, 2 KL1 treated (2x24 weeks) and 3 CKD vehicle treated (1x22 weeks, 2x23 weeks) designated rats spontaneously expired or were euthanized early due to moribundity. During the treatment period, 1 KL1 (age 30 weeks) and 2 CKD rats (ages 29 and 32 weeks spontaneously expired or were euthanized early due to moribundity. The final total of male rats included in the study was 43 (KL1 n=17, CKD n=15, WT n=ll).

[0090] CKD rats developed significant interstitial and perivascular fibrosis compared to WT rats (P<0.0001), which was reduced with KL1 (Fig. 3A). KL1 rats had reduced total fibrotic area in left ventricular (LV) sections (median [min, max range]: 33.45% [19.58%, 39.79%]) compared to CKD control rats (44.90% [41.06%, 52.99%]; P<0.01) as quantified by Masson’s Trichrome staining (Fig. 3B). There was no difference in LVMI (P=0.99) or body weight (P=0.83) between KL1 and CKD rats (Figs. 3C-3D). To further validate anti-fibrotic effects of KL1, applicantassessed LV expression of the pro-fibrotic cytokine, TGF-0 (Figs, 4A-4B). KL1 rats exhibited reduced TGF-P protein expression compared to CKD rats (P=0.015), with expression levels similar to normal littermates (P=0.55).

[0091] To investigate whether KLl’s anti -fibrotic effects involved alterations in myofibroblast differentiation and collagen metabolism, applicant next assessed a-smooth muscle actin (a-SMA), metalloproteinases (MMPs), and collagen cross-linking by lysyl hydroxylase (LOX, Fig.4C). KL1 decreased a-SMA compared to both CKD rats (P=0.003) and WT rats (P=0.008, Fig.4D). There was no difference in a-SMA between CKD and WT rats (P=0.51). Additionally, there was no difference in collagenases, MMP1 (P=0.76, Fig. 4E) and MMP9 (P=0.71, Fig. 4F), or collagen cross-linking enzyme, LOX (P=0.61, Fig. 4G), across all groups. Applicant also investigated if KLl’s anti-fibrotic effects involved reduction in cardiac inflammation, and found no difference in inflammatory markers across all groups (P>0.05, Figure 9).Anti -fibrotic effects of KL1 did not involve alterations in renal function, in vivo.

[0092] Anti-fibrotic effects of KL1 involving alterations in kidney function were investigated by assessing rat eGFR (based on BUN, creatinine, and body weight) and renal histology. As expected, plasma BUN (P<0.0001, Fig. 5 A) and serum creatinine (P=0.0001, Fig. 5B) were increased in CKD rats compared to WT rats. However, neither BUN (P=0.33) nor creatinine (P=1.0) in KL1 rats were significantly different compared to CKD rats. In turn, rat eGFR was not altered in KL1 treated rats (0.415 mL / min [0.105, 0.967]) and remained similar to CKD rats (0.459 mL / min [0.143,1.944], P 0.92) compared to WT rats (2.500 mL / min [1.326, 3.288], P<0.0001, Fig. 5C). Total kidney weight normalized to body weight, a surrogate measurement for cystic development (P=0.58, Fig. 5D), and total fibrotic area as assessed by Masson’s Trichrome staining (P=0.19, Figs. 5E-5F) were similar between KL1 and CKD rats.Anti-fibrotic effects of KL1 did not involve alterations in mineral metabolism, in vivo.

[0093] To investigate whether anti-fibrotic effects of KL1 involve modulations in mineral levels and hormonal regulators, circulatory levels of inorganic phosphorous (Pi), calcium, intact (iFGF23) and C-terminus (cFGF23, representing total FGF23), and PTH were assessed. CKD rats had elevated plasma Pi (10.45 mg / dL [6.73, 18.39]) compared to WT rats (5.56 mg / dL [3.72, 9.84], P<0.0001, Fig. 6A). KL1 (10.45 mg / dL [6.73, 18.49]) did not reduce Pi compared to CKDrats (P=l .0). Plasma calcium was similar across all groups (P=0.18, Fig. 6B). CKD rats had elevated iFGF23 (7.24 ng / mL [1.27, 32.00]) compared to WT rats (0.63 ng / mL [0.15, 1.05], P<0.0001, Fig. 6C). However, KL1 (12.05 ng / mL [1.9, 36.25]) did not significantly alter iFGF23 compared to CKD rats (P=0.48). Similarly, cFGF23 was higher in CKD (10.55 ng / mL [0.78, 40.35]) rats compared to WT rats (0.68 ng / mL [0.15, 2.03], P<0.0001), but not compared to KLl-treated rats (10.86 ng / mL [1.9, 40.03], P=0.63, Fig. 6D). Intact PTH (iPTH) was elevated in CKD (2.49 ng / mL [0.52, 5.67]) rats compared to WT rats (0.15 ng / mL [0.13, 0.22], P<0.0001, Fig. 6E). There was no difference in iPTH between KL 1 (2.51 ng / mL [0.72, 10.25]) and CKD rats (P=0.97).

[0094] KL1 directly inhibits collagen deposition and prevents transformation of cardiac fibroblasts into a secretory myofibroblast phenotype, in vitro.

[0095] To investigate if KL1 has a direct effect on cardiac fibroblasts, the primary drivers of fibrogenesis, an established in vitro cardiac fibrosis model for CKD was utilized and human adult ventricular fibroblasts (HAVFs) were treated with either KL1 or vehicle. High phosphate and calcium-exposed HAVFs (“P+C”) elevated collagen type I (COL1, P<0.0001) compared to unexposed HAVFs (“NOR”, Fig. 7A-B). However, KL1 decreased COL1 (P<0.0001) compared to vehicle controls (“P+C”). There was no statistical difference in COL1 between KL1 and NOR HAVFs (P=0.54). Collagen type III (COL3) did not differ across all groups (P=0.5, Fig. 7C). In turn, KL1 HAVFs exhibited reduced COL1 / 3 ratio versus P+C HAVFs (P=0.0003) to levels like NOR HAVFs (P=0.69, Fig. 7D).

[0096] KLl’s antifibrotic effects were investigated to determine if they involve the regulation of myofibroblast differentiation and collagen regulation of HAVFs. a-SMA was reduced in P+C compared to NOR HAVFs (P=0.012), and further in KL1 compared to P+C HAVFs (P=0.014, Fig. 7E). There was no difference in MMP1 across all groups (P=0.19, Fig. 7F). KL1 also helped preserve a spindle-like, non-secretory morphology as seen in NOR HAVFs compared to P+C HAVFs (Fig. 7G).

[0097] Animal studies have demonstrated that administration of exogenous Klotho can help preserve renal function in acute kidney injury and prevent progression into CKD. Reno protective effects have also been described with exogenous KL1. Interestingly, Applicant foundthat KL1 did not alter BUN, creatinine, normalized total kidney weight, a surrogate measure of renal cysts, nor renal fibrosis. In contrast, Takenaka et al. demonstrated that KL1 in DBA / 2-pcy mice, an autosomal recessive Polycystic Kidney Disease (PKD) model35, improved GFR, mean blood pressure, and slowed progression of renal cyst development and renal fibrosis.23 In this study, applicant utilized the Cy / + cystic rat model, which involves an autosomal dominant non-orthologous mutation, and is well-established to develop a human-like CKD-MBD phenotype. The difference in KLl’s renoprotective effect observed between these studies could be due to several reasons: First, a greater mass effect of renal cysts and CKD-MBD phenotype severity may be observed in the Cy / + rat versus DBA / 2-pcy mice. Additionally, Takenaka et al. started daily lOpg / kg KL1 injections in DBA / 2-pcy mice at 6 weeks-old which is 12 weeks younger than the timepoint when azotemia starts to develop in DBA / 2-pcy mice. Our group started daily 50pg / kg KL1 injections in Cy / + rats at 27 weeks-old which is 17 weeks after azotemia starts to develop. An earlier KL1 dosing regimen or a higher dosage for advanced stages of CKD in our model may unmask renoprotective effects. Nevertheless, the negligible improvement in renal function with amelioration in cardiac fibrosis in our study suggests that KL1 can exert cardioprotective effects independently of improvements in kidney function.

[0098] Summary of the direct anti -fibrotic effects of KL1 in CKD-MBD. KL1 treatment decreased cardiac fibrosis by decreasing collagen type I, TGF-P, and u.-SMA without any changes in collagen type III, collagenases MMP1 and MMP9, or collagen cross-linking enzyme LOX. Downregulation of a-SMA suggests KL1 ameliorates the pro-fibrotic myofibroblast phenotype to reduce fibrosis as evidenced by decreased collagen type I and TGF-p. These anti-fibrotic effects of KL1 did not involve alterations in renal function (i.e., changes in BUN, creatinine, renal cysts and fibrosis) or mineral metabolism (i.e., changes in Pi [or renal clearance of Pi], Ca2+, PTH, or FGF23).

Claims

Claims1. A method for reducing cardiac fibrosis and / or vascular smooth muscle cell calcification in a patient in need thereof, said method comprising administering to said patient a pharmaceutical composition comprising a KL1 peptide in an amount effective to decrease one or more of collagen type I, TGF-P, and a-SMA levels in cardiac fibroblast cells, and / or reduce vascular smooth muscle cell calcification in said patient.

2. The method of claim 1 wherein the patient has Chronic Kidney Disease.

3. The method of claim 1 or 2 wherein said KL1 peptide comprises at least a 100 amino acid N-terminal fragment of SEQ ID NO: 1.

4. The method of claim 3 wherein said KL1 peptide comprises an amino acid selected from the group consisting of SEQ ID NO: 2, or a peptide that differs from SEQ ID NO: 2, by one or two amino acid substitutions.

5. The method of claim 3 wherein said KL1 peptide comprises an amino acid having at least 95% sequence identity to SEQ ID NO: 2.

6. The method of claim 1 wherein said KL1 peptide is acylated with an acyl group comprising a C16-C18 fatty acid or C16-C18 fatty diacid chain.

7. The method of claim 1 wherein said peptide is administered parenterally.

8. The method of claim 1 wherein said peptide is administered via intravenous injection.

9. A method of treating a patient to reduce cardiac fibrosis and / or vascular smooth muscle cell calcification, said method comprisingidentifying a patient having, or at risk of cardiac fibrosis or vascular smooth muscle cell calcification;administering to said identified patient a pharmaceutical composition comprising a KL1 peptide, wherein said peptide decreases collagen type I, TGF-P, and / or a-SMA concentrations in human cardiac fibroblast cells upon contact of said fibroblast cells with said KL1 peptide.

10. The method of claim 9 wherein said patient is afflicted with Chronic Kidney Disease (CKD) and the method comprises the step of first identifying a patient with CKD.

11. The method of claim 9 wherein said KL1 peptide comprises at least a 100 amino acid N-terminal fragment of SEQ ID NO: 1.

12. The method of claim 9 wherein said KL1 peptide comprises an amino acid selected from the group consisting of SEQ ID NO: 2, or a peptide that differs from SEQ ID NO: 2, by one or two amino acid substitutions.

13. The method of claim 9 wherein said KL1 peptide comprises an amino acid having at least 95% sequence identity to SEQ ID NO: 2.

14. The method of claim 9 wherein said KL1 peptide is acylated with an acyl group comprising a C16-C18 fatty acid or C16-C18 fatty diacid chain.

15. A pharmaceutical composition comprising a KL1 peptide, wherein said peptide decreases collagen type I, TGF-0, and / or a-SMA concentrations in human cardiac fibroblast cells upon contact of said fibroblast cells with said KL1 peptide.

16. The composition of claim 15 wherein said KL1 peptide comprises at least a 100 amino acid fragment of SEQ ID NO: 2.

17. The composition of claim 15 wherein said KL1 peptide comprises an amino acid selected from the group consisting of SEQ ID NO: 2, or a peptide that differs from SEQ ID NO: 2, by one or two amino acid substitutions.

18. The composition of claim 15 wherein said KL1 peptide comprises an amino acid having at least 95% sequence identity to SEQ ID NO: 2.

19. A peptide dimer comprising a first and second peptide independently selected from the peptides of claim 3, wherein the first and second peptides are covalently linked via a linker.

20. The composition of claim 15 wherein said KL1 peptide is acylated with an acyl group comprising a C16-C18 fatty acid or C16-C18 fatty diacid chain.

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