IL-6 inhibitors as treatments for nephropathy

Abstract

Methods and compositions are provided for treating diseases or disorders (eg, organ fibrosis, nephropathy) by administering inhibitors of IL-6 activity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 313,101, filed February 23, 2022, the disclosure of which is incorporated herein by reference in its entirety. GOVERNMENT SUPPORT

[0002] This invention was made with government support under HL131952 awarded by the National Institutes of Health. The United States Government has certain rights in this invention. [Technical field]

[0003] The present disclosure relates generally to the use of IL-6 inhibitors for the treatment of diseases and disorders such as organ fibrosis or nephropathy.

[0004] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format, and is incorporated by reference in its entirety. The XML copy created on February 20, 2023 has the filename 251609_000088_SL.XML and is 23,686 bytes in size. [Background technology]

[0005] Approximately one-third of diabetic patients develop diabetic nephropathy (DN), which is the leading cause of end-stage renal disease (ESRD), causing more than 950,000 deaths worldwide each year (Non-Patent Documents 1, 2). 1、2 Over the past 20 years, no new drugs have been approved to specifically prevent diabetic nephropathy or improve renal function (Non-Patent Document 3). 3 This lack of progress is due, in part, to a poor understanding of the mechanisms underlying progressive renal dysfunction. Furthermore, this knowledge gap contributes to the suboptimal treatment options available to these patients. Improved understanding of the mechanisms underlying diabetic kidney disease pathogenesis is urgently needed to facilitate the development of novel, effective, and safe therapeutic agents that can target the early stages of diabetes before renal damage becomes irreversible.

[0006] Diabetic nephropathy is characterized by excessive deposition of extracellular matrix, loss of capillary networks, and accumulation of fibrillar collagen, activated myofibroblasts, and inflammatory cells (Non-Patent Documents 4, 5). 4、5 In renal fibrosis, myofibroblasts are thought to be a phenotype of activated fibroblasts that contribute to fibrosis (Non-Patent Documents 5 and 6). 5、6 There are six well-reported sources of matrix-producing myofibroblasts: (1) activated resident fibroblasts, (2) differentiated pericytes, (3) recruited circulating fibrocytes, (4) from macrophages via macrophage-to-mesenchymal transition (5), from mesenchymal cells derived from renal tubular epithelial cells via epithelial-to-mesenchymal transition (EMT), and (6) from mesenchymal cells transformed from endothelial cells (ECs) via endothelial-to-mesenchymal transition (EndMT) (Non-Patent Documents 7, 8). 7、8 .

[0007] Among these diverse sources of matrix-producing fibroblasts, mesenchymal cells transformed from ECs via EndMT (Non-Patent Documents 7, 8) 7、8 is an important source of myofibroblasts in several organs, including the kidney (Non-Patent Document 9). 9 EndMT is characterized by the loss of endothelial markers such as cluster of differentiation 31 (CD31) and the acquisition of expression of mesenchymal proteins such as α-smooth muscle actin (αSMA), vimentin, and fibronectin (Non-Patent Documents 7, 8). 7、8 .

[0008] ECs are important contributors to neovascularization in health and life-threatening diseases (Non-Patent Document 10). 10 Disruption of EC central metabolism contributes to disease phenotypes (Non-Patent Documents 11, 12). 11、12Carnitine palmitoyltransferase 1a (CPT1a)-mediated fatty acid oxidation (FAO) regulates the proliferation of ECs in the stalks of sprouting blood vessels (Non-Patent Documents 13-15). 13-15 ECs use metabolites / precursors for epigenetic control of their subtype differentiation and maintain crosstalk via metabolites released by other cell types (10, 15). 10、15 In particular, EndMT induces changes in endothelial cell metabolism and is an area of ​​active research (Non-Patent Documents 16, 17). 16、17 For example, mesenchymal cells derived from EndMT reprogram their metabolism and exhibit defective fatty acid metabolism (Non-Patent Document 17). 17 .

[0009] The contribution of EndMT to renal fibrosis has been analyzed in several mouse models of chronic kidney disease (Non-Patent Documents 5-7, 18, 19). 5-7、18、19 Zeisberg et al. conducted seminal experiments and reported that in the kidneys of mice with unilateral ureteral obstructive nephropathy (UUO), approximately 30-50% of fibroblasts co-express the EC marker CD31 along with fibroblast and myofibroblast markers such as fibroblast-specific protein 1 (FSP-1) and αSMA (Non-Patent Document 19). 19 Complete conversion of ECs to mesenchymal cell types is not necessary, as intermediate cell types are sufficient to trigger the activation of profibrogenic pathways. EndMT can induce profibrogenic signaling in neighboring cells by autocrine and / or paracrine mechanisms, thereby contributing to overall renal fibrosis (6, 20, 21). 6、20、21 Therefore, targeting EndMT may have therapeutic potential for the treatment of renal fibrosis (Non-Patent Documents 6, 19, 22). 6、19、22 . [Prior art documents] [Non-patent literature]

[0010] [Non-licensed document 1] Reidy, K. et al., J Clin Invest 124, 2333-2340, (2014)

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[0011] The glucocorticoid receptor (GR) is a nuclear hormone receptor that is ubiquitously expressed in most cell types and is important in many conditions of health and disease. The glucocorticoid receptor (GR) mediates the actions of steroid hormones in various tissues, including the kidney. The role of glucocorticoids in cardiovascular and renal disease is complex. The inventors of the present disclosure have demonstrated that GR mediates the actions of steroid hormones in the treatment of cardiovascular and renal diseases such as sepsis. 23 and atherosclerosis 25 identified endothelial GR as a negative regulator of vascular inflammation in a model of renal fibrosis. Loss of endothelial GR can lead to upregulation of the canonical Wnt signaling pathway. This pathway is also upregulated in renal fibrosis. However, it is unclear whether endothelial GR contributes to the regulation of fibrogenic processes in the progression of renal fibrosis.

[0012] Based on the research presented herein, we show that mice lacking endothelial GR accelerate renal fibrosis in both diabetic and non-diabetic conditions. This exacerbation of fibrosis is associated with abnormal chemokine and cytokine reprogramming, enhanced Wnt signaling, and suppressed fatty acid oxidation. This disclosure demonstrates that endothelial GR is a key molecule involved in the regulation of fibrotic processes in the kidney.

[0013] One aspect of the present disclosure provides a method for treating a condition or disease in a subject that requires treatment.The method may comprise administering a therapeutically effective amount of an inhibitor of IL-6 activity to the subject, and the condition or disease is selected from organ fibrosis, nephropathy, dyslipidemia, hypertension, hyperlipidemia, hypercholesterolemia, cardiovascular disease, peripheral artery disease, atherosclerosis, coronary artery disease, coronary heart disease, and stroke.In one embodiment, the organ fibrosis is renal fibrosis.In one embodiment, the nephropathy is diabetic nephropathy.

[0014] In some embodiments of the treatment methods described herein, the inhibitor of IL-6 activity is an antibody or an antigen-binding fragment of an antibody. In some embodiments, the inhibitor is an IL-6 neutralizing antibody or an antigen-binding fragment thereof, such as, but not limited to, sirukumab, siltuximab, or olokizumab, or an antigen-binding fragment thereof. In some embodiments, the inhibitor is an antibody or an antigen-binding fragment that targets the IL-6 receptor, such as, but not limited to, tocilizumab or sarilumab, or an antigen-binding fragment thereof.

[0015] In some embodiments of the treatment methods described herein, the inhibitor of IL-6 activity results in blocking trans-signaling, such as, but not limited to, olamkicept (FE999301), or a fragment or variant thereof.

[0016] In some embodiments of the treatment methods described herein, the inhibitor of IL-6 activity is an inhibitor of intracellular signaling, such as, but not limited to, tofacitinib or CpG-stat3 siRNA.

[0017] In various embodiments of the methods of treatment described herein, the methods further comprise administering to the subject one or more additional agents effective to treat said condition or disease.

[0018] In some embodiments, the one or more additional agents include one or more cholesterol-lowering agents, blood pressure-lowering therapies, anti-inflammatory agents, antithrombotic agents, anticoagulants, renin-angiotensin-aldosterone system inhibitors (RAAS inhibitors), beta-adrenergic blockers, calcium channel blockers, hypoglycemic agents, and / or Wnt inhibitors.

[0019] In some embodiments, cholesterol-lowering drugs include statins, fibrates, and / or inhibitors of proprotein convertase subtilisin / kexin type 9.

[0020] In some embodiments, the antihypertensive therapy comprises an angiotensin-converting enzyme (ACE) inhibitor and / or an angiotensin II receptor blocker (ARB).

[0021] In some embodiments, the hypoglycemic agent comprises metformin, insulin, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide 1 (GLP-1), and / or a sodium / glucose co-transporter 2 (SGLT2) inhibitor.

[0022] In some embodiments, the Wnt inhibitor comprises a compound having the structure of Formula (I), or a pharma- ceutically acceptable salt thereof: [ka] [In the formula, X1 and X2 are selected from N and CR; one of X3, X4, X5 and X6 is N, and the others are selected from N and CR; X7, X8, X9 and X 10 one of is N and the others are selected from N and CR; X 11 , X 12 , X 13 and X 14 one of is N and the others are selected from N and CR; R is independently at each occurrence selected from hydrogen, halo, cyano, methyl, difluoromethyl, and trifluoromethyl.

[0023] In various embodiments of the treatment methods described herein, the subject is a human. [Brief description of the drawings]

[0024] [Figure 1] Figures 1A-1B show that loss of endothelial glucocorticoid receptor (GR) leads to a fibrogenic phenotype in the kidneys of diabetic mice. (Figure 1A) Immunofluorescence analysis was performed in kidneys of control and diabetic CD-1 and C57BL / 6 mice using FITC-labeled GR, rhodamine-labeled CD31, and DAPI. Representative merged images are shown; original magnification 400x. Scale bar 50 µm. N=7 biologically independent mice / group combined from three separate experiments. Quantification of GR / CD-31 double-labeled cells is shown. Data are mean ± SEM. (Figure 1B) Western blot and qPCR analysis of GR protein and mRNA levels in endothelial cells isolated from kidneys of control and diabetic CD-1 and C57BL / 6 mice. Endothelial cells were isolated and cultured from six biologically independent mice / group. Three independent experiments were performed. Densitometry analysis of six samples / group is shown and normalized to β-actin. mRNA expression from six independent cell culture samples was analyzed and normalized to 18S. Two independent experiments were performed in triplicate. Data are means ± SEM. For all panels, two-way ANOVA was used to analyze statistical significance. Significance: *p<0.05, **p<0.01, ***p<0.001. [Figure 2-1]Figures 2A-2J show that loss of endothelial GR exacerbates renal fibrosis in diabetic mice. (Figure 2A) Schematic diagram showing induction of diabetes in GR fl / fl;Tie1 Cre- (control), GR fl / fl;Tie1 Cre+ (GRECKO), Apoe- / -;GR fl / fl;Tie1 Cre- (Apoe- / -) and Apoe- / -;GR fl / fl;Tie1 Cre+ (DKO) mice. Diabetes and renal fibrosis were induced by five injections of STZ (50 mg / kg / day IP). (Figure 2B-2I) Physiological parameters such as body weight, blood glucose (blood glucose level), kidney weight / body weight, heart weight / body weight, liver weight / body weight, albumin / creatinine ratio (ACR), plasma triglycerides and plasma cholesterol were measured. N=6 biologically independent mice / group obtained from two separate experiments. [Figure 2-2] (FIG. 2J) Masson trichome and Sirius red staining in kidneys of non-diabetic and diabetic control, GRECKO, Apoe- / - and DKO mice were analyzed. Representative images are shown; original magnification 300x. Relative area of ​​fibrosis (% fibrosis area) and relative collagen (%) were measured using ImageJ program. N=7 biologically independent mice / group from two separate experiments. Scale bar 50 μm. Data are mean ± SEM. For all panels, two-way ANOVA was used to analyze statistical significance. Significance: *p<0.05, **p<0.01, ***p<0.001. [Diagram 3]Figures 3A-3C show that neutralization of IL-6 rescues renal fibrosis in diabetes. (Figure 3A) Plasma IL-6 levels in nondiabetic and diabetic mice were measured by cytokine array (Luminex). N=5 biologically independent mice / group. (Figure 3B) Measurement of IL-6 in the medium of cultured endothelial cells isolated from the indicated groups. Endothelial cells were isolated and cultured from 6 biologically independent mice per group and analyzed in triplicates. Data are mean ± SEM. (Figure 3C) Neutralization of IL-6 in diabetic GRECKO and DKO mice. Representative images are shown; original magnification 300x. Relative fibrosis area (%) and relative collagen (%) were measured using the ImageJ program. N=7 biologically independent mice / group. Scale bar 50 μm. Data are mean ± SEM. For all panels, one-way ANOVA with Tukey post-hoc test was used to analyze statistical significance. *p<0.05, **p<0.01, ***p<0.001. [Figure 4-1]Figures 4A-4C show upregulation of Wnt signaling and fibrogenic markers by loss of endothelial GR. (Figure 4A) Western blot analysis of vimentin, HIF1α, Snail1, active β-catenin, CPT1a and PPARα in endothelial cells isolated from kidneys of non-diabetic and diabetic control and GRECKO mice. Endothelial cells were isolated and cultured from six biologically independent mice / groups. Densitometry calculations from six samples / groups combined from three independent experiments are shown and normalized to β-actin or HSP90 as indicated. Representative blots are shown. Data are mean ± SEM. (Figure 4B) Western blot analysis of αSMA, fibronectin, active β-catenin and β-catenin in endothelial cells isolated from kidneys of diabetic control, GRECKO, Apoe- / - and DKO mice. Endothelial cells were isolated and cultured from six biologically independent mice / groups. Densitometry calculations from six samples / groups combined from three independent experiments are shown and normalized to β-actin or HSP90 as indicated. Data are means ± SEM. Representative blots are shown. [Figure 4-2] (Figure 4C) Immunofluorescence analysis of αSMA / CD31 and TGFβR1 / CD31 was performed in kidneys of non-diabetic and diabetic control, GRECKO, Apoe- / - and DKO mice. FITC-labeled αSMA, FITC-labeled TGFβR1, rhodamine-labeled CD31, and DAPI (nuclei) were used. Representative merged images are shown; original magnification 300x. Scale bar 50 μm. N=7 biologically independent mice / group. Data are mean ± SEM. For all panels, one-way ANOVA with Tukey post-hoc test was used to analyze statistical significance. Significance: *p<0.05, **p<0.01, ***p<0.001. [Diagram 5]Figure 5A-5C show that Wnt inhibitors partially suppress renal fibrosis in diabetic GRECKO and DKO mice. (Figure 5A) Schematic showing the treatment protocol of Wnt inhibition in diabetic control and GRECKO mice. (Figure 5B) Physiological parameters including body weight, blood glucose (blood glucose level) and kidney weight / body weight were analyzed. N=6 biologically independent mice / group combined from two experiments. (Figure 5C) Immunohistochemical analysis of Masson's trichrome staining, Sirius red staining and PAS staining as well as β-catenin levels in kidneys of wnti-treated diabetic control and GRECKO mice. Representative images are shown; original magnification 300x. Relative fibrosis area (%) and relative collagen deposition (%) were measured using the ImageJ program. N=8 biologically independent mice / group combined from three experiments were analyzed. Scale bar 50 μm. Data are mean ± SEM. For all panels, one-way ANOVA with Tukey post-hoc test was used to analyze statistical significance. Significance: *p<0.05, **p<0.01, ***p<0.001. [Figure 6-1]Figures 6A-6E show that metabolic reprogramming by loss of endothelial GR exacerbates diabetic kidney disease. (Figure 6A) Radiolabeled 14C-palmitate uptake analysis in endothelial cells isolated from kidneys of non-diabetic and diabetic control, GRECKO, Apoe- / - and DKO mice. CPMs for each sample were counted. Data were normalized to μg protein. Activity folds are shown. Endothelial cells isolated and cultured from 6 biologically independent mice / group for 14C-palmitate uptake analysis were experimented and analyzed in triplicates. Data are mean ± SEM. (Figure 6B) Radiolabeled [14C]palmitate oxidation and [14CO2] release were measured in endothelial cells isolated from kidneys of non-diabetic and diabetic control, GRECKO, Apoe- / - and DKO mice. CPMs for each sample were counted. Data were normalized to μg protein. Activity folds are shown. Endothelial cells isolated and cultured from 6 biologically independent mice / group for 14C-palmitic acid uptake analysis were analyzed in triplicate experiments. Data are mean ± SEM. (Figure 6C) At the end of these experiments, body weight, blood glucose (blood glucose level), and kidney weight / body weight were measured. N=6 biologically independent mice from 2 experiments. Ex vivo oxidation of radiolabeled [14C]palmitic acid and [14CO2] release were measured in the kidney. CPM of each sample were counted. Endothelial cells isolated from 5 biologically independent mice / group were analyzed in triplicate. Data are mean ± SEM. [Figure 6-2](Fig. 6D) Masson's trichrome staining, Sirius red staining and PAS staining in kidneys of control, etomoxir-treated and C75-treated diabetic mice were analyzed. Representative images are shown; original magnification 300x. Relative fibrosis area (%) and relative collagen deposition (%) were measured using the ImageJ program. N=8 biologically independent mice per group from three independent experiments were analyzed. Scale bar 50 μm. Data are mean ± SEM. (Fig. 6E) Immunohistochemical analysis of CPT1a and β-catenin. Representative images are shown; original magnification 300x. Scale bar 50 μm. N=6 biologically independent mice per group from three independent experiments. For all panels, one-way ANOVA with Tukey post-hoc test was used to analyze statistical significance. Significance: *p<0.05, **p>0.01, ***p<0.001. [Figure 7-1]Figures 7A-7J show that GR loss in endothelial cells reprograms central metabolism in renal tubular cells. (Figure 7A) Conditioned medium experimental design. HUVECs were transfected with scrambled or GR siRNA; after 6 h, medium was replaced and cells were incubated for 96 h. The collected medium was then transferred to HK-2 cells. (Figure 7B) Representative Western blot analysis of E-cadherin, αSMA, TGFβR1 and active β-catenin expression. N=7 independent experiments. Representative blots from 7 biologically independent samples / groups were combined from 4 independent experiments. Densitometry analysis values ​​are shown relative to β-actin. (Figure 7C) Immunofluorescence analysis of E-cadherin and α-SMA expression in conditioned medium-treated TECs. For each slide, images of 6 different fields at Å-400 magnification were evaluated. n=7 independent samples / group. Scale bar 30 μm. (Figure 7D) Oxidation of [14C]palmitate as measured by [14CO2] release. CPM were counted and normalized to protein in the well. (Figure 7E) Oxygen consumption rate (OCR) in conditioned medium-treated TECs; each data point represents the average of eight independent samples. Three independent experiments were performed. OCR was measured on a Seahorse XF96 analyzer. (Figure 7F) Cellular ATP measurement. Eight independent samples were analyzed. (Figure 7G) Relative mRNA levels determined by qRT-PCR of fatty acid oxidation (FAO) regulators in conditioned medium-treated TECs. [Figure 7-2](Figure 7H) Experimental design for conditioned medium experiments. ECs from kidneys of diabetic GRECKO and diabetic control mice were cultured for 96 h. The collected medium was then transferred to TECs from diabetic control mice. n=7 / group. (Figure 7I) Representative western blot analysis of expression of αSMA, TGFβR1, active β-catenin, E-cadherin, CPT1a and PPARα. Representative blots from seven biologically independent samples / group combined from three independent experiments are shown. Densitometry calculations are normalized to β-actin or HSP90 as indicated. (Figure 7J) Relative mRNA levels determined by qRT-PCR of regulators of FAO in conditioned medium-treated isolated TECs. Data are mean ± SEM. Student's t-test (unpaired, two-tailed) was used for Figures 7B-7F; Figure 7I; for Figures 7G and 7J, one-way ANOVA with Tukey post-hoc test was used to analyze statistical significance. Significance: *p<0.05, **p<0.01, *** / p<0.001. [Figure 8] A diagrammatic representation of the role of GR in endothelial cell homeostasis is shown. GR is crucial for endothelial cell homeostasis, and its loss causes disruption of homeostasis through the release of the proinflammatory cytokine IL-6 and aberrant activation of Wnt signaling, which leads to defective fatty acid oxidation and associated mesenchymal activation. [Figure 9]Figures 9A-9C show the effect of adrenalectomy on renal fibrosis. Adrenalectomized and non-adrenalectomized control and diabetic mice of CD-1 and C57BL / 6 strains were evaluated 4 weeks after bilateral adrenal surgery. (Figure 9A) Measurement of plasma corticosterone in control and diabetic CD-1 and C57BL / 6 mice. n=7 biologically independent mice / group combined from two separate experiments. (Figure 9B) Blood glucose measurement in adrenalectomized and non-adrenalectomized mice. n=7 biologically independent mice / group combined from two separate experiments. (Figure 9C) Masson's trichrome and Sirius red staining in kidneys of adrenalectomized and non-adrenalectomized control and diabetic mice. Representative images are shown; original magnification 300x. Fibrosis area (%) and relative collagen deposition (%) were measured using the ImageJ program. n=7 biologically independent mice / group combined from two separate experiments. Scale bar 50 μm. Data are means ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey's multiple comparison post-hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. C-CD1: non-diabetic control CD1 mice, DM-CD1: diabetic CD-1 mice, C-BL6: non-diabetic control C57BL / 6 mice, DM-BL6: diabetic C57BL / 6 mice, and ADX: adrenalectomy. [Figure 10] Determination of kidney endothelial cell purity. Top: Flow cytometry cell purity analysis in endothelial cells isolated from kidney. CD31+CD45- cells are shown. Bottom: Gating strategy in unstained blocked endothelial cells. [Figure 11]Figures 11A-11B show the analysis of GR protein and mRNA levels in isolated endothelial cells. (Figure 11A) Western blot analysis of GR protein levels and (Figure 11B) qPCR analysis of GR mRNA expression were analyzed in endothelial cells isolated from kidneys of control and GRECKO mice. n=7 biologically independent control mice, n=8 biologically independent GRECKO mice. Three independent experiments were analyzed. Data are means ± SEM. Student's t-test (unpaired, two-tailed) was used to analyze statistical significance. Significance: *p<0.05, **p<0.01, ***p<0.001. [Figure 12] Physiological characteristics of non-diabetic mice. Body weight, blood glucose, kidney / body weight, heart / body weight, liver / body weight, albumin / creatinine ratio, plasma triglycerides, plasma cholesterol and systolic blood pressure are shown in non-diabetic control, GRECKO, ApOe- / - and DKO mice. n=6 biologically independent mice / group (blood pressure n=8 biologically independent mice / group) were combined from two separate experiments. Data are mean ± SEM. Statistical significance was calculated using One-way ANOVA with Tukey's multiple comparison post-hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. [Figure 13] Figures 13A and 13B show the assessment of glomerular fibrosis in non-diabetic and diabetic mice. (Figure 13A) Periodic acid Schiff (PAS) staining in kidneys of non-diabetic and diabetic control, GRECKO, Apoe- / - and DKO mice. Scale bar 50 μm. n=7 biologically independent mice / group combined from two separate experiments. Representative images are shown. (Figure 13B) Immunohistochemical analysis of vimentin expression in kidneys of non-diabetic and diabetic control, GRECKO, Apoe- / - and DKO mice. n=7 biologically independent mice / group combined from two separate experiments. Scale bar 50 μm. Representative images are shown; original magnification 200x. [Figure 14]Expression of Snail1, PPARα and HIF1α in non-diabetic and diabetic mice. Immunohistochemical analysis of Snail1, PPARα and HIF1α expression in kidneys of non-diabetic and diabetic control, GRECKO, Apoe- / - and DKO mice. Scale bar 50 μm. n=7 biologically independent mice / group combined from two separate experiments. Representative images shown. Original magnification 200x. [Figure 15] Collagen I and fibronectin deposition in non-diabetic and diabetic mice. Immunofluorescence analysis of collagen I and fibronectin was performed in kidneys of non-diabetic and diabetic control, GRECKO, Apoe- / - / and DKO mice. FITC-labeled collagen I, rhodamine-labeled fibronectin, and DAPI were used. Scale bar 50 μm. n=7 biologically independent mice / group. Representative images from two separate experiments are shown; original magnification 300x. [Figure 16]Figures 16A-16C show that loss of endothelial GR exacerbates fibrosis in a mouse model of unilateral ureteral obstruction (UUO). (Figure 16A) Schematic of the UUO model. The left kidney of control littermates and GRECKO mice was ligated. The kidneys were removed on days 5 and 10. (Figure 16B) Masson's trichrome and Sirius red staining in the contralateral kidney and kidney subjected to UUO surgery were analyzed. Representative images are shown; original magnification 200x. Fibrosis area (%) and relative collagen deposition (RCD%) were measured using the ImageJ program. n=8 biologically independent mice / group combined from three independent experiments. Scale bar 50 μm. Data are mean ± SEM. Statistical significance was calculated using two-way ANOVA with Tukey's multiple comparison post-hoc test. Significance: *p<0.05. (FIG. 16C) Immunofluorescence analysis of collagen I, aquaporin / α-SMA, and aquaporin / fibronectin was performed in the contralateral and UUO-operated kidneys of control and GRECKO mice. FITC-labeled collagen I, α-SMA, and fibronectin, rhodamine-labeled aquaporin, and DAPI were used. Representative images are shown; original magnification 300x. Scale bar 50 μm. n=7 biologically independent mice / group combined from two experiments. Con: control; Contra: contralateral kidney; FN: fibronectin. [Figure 17]17A-17B show dexamethasone treatment (Dexa) in urinary tract obstruction. (FIG. 17A) Masson's trichrome staining in dexamethasone-treated and vehicle-treated UUO mice; original magnification 200x. Mice were treated with dexamethasone 1 mg / kg in drinking water until the day of sacrifice. Time-dependent analysis on days 10 and 15 is shown. Representative images are shown. Relative fibrosis area (%) was measured using ImageJ program. n=7 biologically independent mice / group combined from two separate experiments. Scale bar 50 μm. Data are mean±SEM. Statistical significance was calculated using one-way ANOVA with Tukey's multiple comparison post-hoc test. Significance: ***p<0.001. (FIG. 17B) Masson's trichrome staining in dexamethasone-treated (Dexa) and vehicle-treated non-diabetic CD-1 mice (original magnification 200x). Representative images are shown. Relative fibrosis area (%) was measured using the ImageJ program. n=6 biologically independent mice / group. Scale bar 50 μm. Data are mean ± SEM. Data were analyzed by Student's s test (two-tailed unpaired test). [Figure 18] We show that diabetic kidney disease is associated with cytokine and chemokine reprogramming. Plasma cytokines and chemokines were measured in non-diabetic control and diabetic CD-1 and C57BL / 6 mice by using cytokine array analysis (Luminex). n=5 biologically independent mice / group. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey multiple comparison post-hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. C-CD1: non-diabetic control CD1 mice, DM-CD1: diabetic CD-1 mice, C-BL6: non-diabetic control C57BL / 6 mice, DM-BL6: diabetic C57BL / 6 mice. [Figure 19]Figures 19A-19B show cytokine and chemokine reprogramming in non-diabetic and diabetic GRECKO and DKO mice. (Figure 19A) Plasma cytokines and chemokines were measured in non-diabetic and diabetic GRECKO and DKO mice by using cytokine array analysis (Luminex). n=5 biologically independent mice / group. Data are mean ± SEM. (Figure 19B) Relative gene expression analysis of the indicated cytokines was evaluated in diabetic kidneys. Expression levels were normalized using 18S. Data are mean ± SEM. In both panels, statistical significance was calculated using one-way ANOVA with Tukey multiple comparison post-hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. [Figure 20-1] Figures 20A-20D show that neutralization of IL-6 rescues the fibrotic phenotype in the kidney of diabetic CD-1 mice. IL-6 IgG and control IgG were injected intraperitoneally at a dose of 3 mg / kg, three times a week for 4 weeks, into both nondiabetic and diabetic CD-1 mice. (Figures 20A-20B) Endothelial cells isolated from CD-1 mice were stimulated with recombinant IL-1β, IL-6, TNFα, and TGFβ, and vimentin and α-SMA were analyzed by Western blot. Representative blots from three independent experiments were analyzed. Densitometric calculations are shown relative to actin. (Figure 20C) Western blot analysis of vimentin in TGFβ-stimulated and non-stimulated vascular endothelial cells treated with N-IL-1β (neutralizing antibody-IL-1β) and N-IL-6 (neutralizing antibody-IL-6) antibodies. Representative blots from three independent experiments were evaluated. Densitometry calculations are shown relative to β-actin. [Figure 20-2](FIG. 20D) Neutralization of IL-6 in control and diabetic CD-1 mice. Representative images of Masson's trichrome and Sirius red staining (original magnification 300x) are shown. n=7 biologically independent mice / group combined from two independent experiments. Relative fibrosis area (%) and relative collagen (%) were measured using the ImageJ program. Scale bar 50 μm. Data are mean ± SEM. In all panels, statistical significance was calculated using one-way ANOVA with Tukey's multiple comparison post-hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. [Figure 21] Neutralization of IL-6 in non-diabetic GRECKO and DKO mice. Both non-diabetic GRECKO and DKO mice were intraperitoneally injected with IL-6 IgG and control IgG at a dose of 3 mg / kg, three times a week for 4 weeks. Representative images (original magnification 300x) of Masson's trichrome staining (top row) and Sirius red staining (bottom row) are shown. n=6 biologically independent mice / group combined from two experiments. Relative fibrosis area (%) and relative collagen (%) were measured using the ImageJ program. Scale bar 50 μm. Data are mean ± SEM. One-way ANOVA with Tukey post-hoc test was used to analyze statistical significance. [Figure 22]Figures 22A-22C show gene expression analysis of Wnt signaling and fibrogenic markers. (Figure 22A) Relative mRNA levels as determined by qRT-PCR of Axin2, Tcf, α-SMA, fibronectin, Snail1, Hif1α and Pparα were analyzed in isolated endothelial cells obtained from kidneys of non-diabetic and diabetic control, GRECKO, Apoe- / - and DKO mice. Endothelial cells from n=6 biologically independent mice / group were evaluated. Combined data from 4 independent experiments were analyzed. Data are mean ± SEM. (Figure 22B) Relative mRNA levels as determined by qRT-PCR of Axin2, Tcf and fibronectin were analyzed in isolated endothelial cells obtained from kidneys of non-diabetic and diabetic control and cultured glomeruli from GRECKO mice. n=6 biologically independent mice / group were analyzed. Combined data from 3 independent experiments were analyzed. Data are mean ± SEM. In (FIG. 22A) and (FIG. 22B), one-way ANOVA with Tukey post-hoc test was used for analysis of statistical significance. Significance: *p<0.05, **p<0.01, ***p<0.001. (FIG. 22C) Immunohistochemical analysis of β-catenin in kidneys of the indicated groups. Representative images shown; original magnification 300×. n=6 biologically independent mice / group combined from 3 independent experiments. Scale bar 50 μm. [Figure 23]Figures 23A-23C show endothelial cell characteristics. (Figure 23A) Endothelial cell leakage was assessed in kidneys of non-diabetic and diabetic control, GRECKO, Apoe- / - and DKO mice 2 hours after mice were injected with fluorescein isothiocyanate (FITC)-dextran. FITC-dextran, rhodamine-labeled CD31, and DAPI were used. Merged images and representative pictures (original magnification 300x) are shown. Arrows indicate cells labeled with FITC-dextran only and areas of leakage. n=7 biologically independent mice / group combined from 3 experiments. Scale bar 50 μm. Quantification is shown below the images. ND: non-diabetic, D: diabetic. Data are mean ± SEM. (Figure 23B) Permeability of isolated endothelial cell monolayers was assessed using Transwell inserts by measuring the passage of FITC-dextran added to the upper compartment into the lower compartment. After 24 hours, the concentration of FITC-dextran in the lower compartment was measured with a fluorescent plate reader. Endothelial cells isolated from 6 biologically independent mice were evaluated. Three independent experiments were analyzed. (Figure 23C) Endothelial cell density (%) measured by CD31 staining in Figure 23A. Endothelial cells isolated from 6 biologically independent mice were evaluated. Three independent experiments were analyzed. Data are mean ± SEM. In all panels, statistical significance was calculated using one-way ANOVA with Tukey's multiple comparison post-hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. [Figure 24]Figures 24A-24J show that inhibition of Wnt signaling abolished the fibrogenic phenotype in mice. (Figure 24A) Immunohistochemical analysis of β-catenin expression in kidneys of control and diabetic CD-1 and C57BL / 6 mice. n=8 biologically independent mice / groups combined from two independent experiments were evaluated. Scale bar 50 μm. Representative images shown; original magnification 200x. (Figure 24B) Relative mRNA expression determined by qRT-PCR of Axin2, Tcf and fibronectin in kidneys of control and diabetic CD-1 and C57BL / 6 mice. n=6 biologically independent mice / groups combined from two experiments were evaluated. Expression was normalized to the internal control 18S. (Figure 24C) Schematic diagram representing the treatment protocol of Wnt inhibitor (LGK974; 5 mg / kg body weight) in diabetic CD-1 mice and (Figure 24D) UUO mice. (Figure 24E) Masson's trichrome staining and Sirius red staining in kidneys of diabetic CD-1 mice and Wnt inhibitor-treated diabetic CD-1 mice. Representative images (original magnification 300x) are shown. Relative fibrosis area (%) and relative collagen deposition (%) were measured using the ImageJ program. n=8 biologically independent mice / groups combined from two experiments were evaluated. (Figure 24F) Masson's trichrome staining and Sirius red staining in kidneys of UUO mice and UUO mice treated with Wnt inhibitors. Representative images (original magnification 300x) are shown. Relative fibrosis area (%) and relative collagen deposition (%) were measured using the ImageJ program. n=8 biologically independent mice / groups combined from two experiments were evaluated. Scale bar 50 μm. (Figures 24G-24H) GR protein levels in CD31-positive cells were analyzed by immunofluorescence in kidneys of Wnti-treated diabetic mice and Wnti-treated UUO mice. FITC-labeled GR, rhodamine-labeled CD31, and DAPI were used. Merged and representative images are shown; original magnification 300×. n=7 biologically independent mice / group combined from 4 experiments were evaluated. Scale bar 50 μm. (Figures 24I-24J) Immunohistochemical analysis of active β-catenin expression in Wnti-treated diabetic and Wnti-treated UUO mice.n=7 biologically independent mice / group combined from 3 experiments were evaluated. Scale bar 30 μm. Data are mean ± SEM. In all panels, statistical significance was calculated using one-way ANOVA with Tukey multiple comparison post-hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. [Diagram 25] Figure 1 shows that inhibition of Wnt signaling prevents cytokine and chemokine reprogramming in diabetic mice. Plasma cytokines and chemokines were measured by using cytokine array analysis (Luminex) in diabetic CD-1 mice with and without Wnti treatment. n=5 biologically independent mice / group. Data are mean±SEM. Student's s test (two-tailed unpaired) was used for statistical significance analysis. Significance: *p<0.05. [Figure 26] Physiological characteristics of Wnt inhibitor treatment in non-diabetic control and GRECKO mice. Physiological parameters including body weight, blood glucose, and kidney / body weight are shown. n=7 biologically independent mice / group combined from two experiments were analyzed. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey multiple comparison post-hoc test. [Figure 27]Figures 27A-27B show histological analysis of kidneys from Wnt inhibitor-treated non-diabetic control mice and GRECKO mice. (Figure 27A) Masson's trichrome staining (MTS) and Sirius red staining in Wnt inhibitor-treated non-diabetic control mice and GRECKO mice. Representative images are shown; original magnification 200x. Fibrosis area (%) and relative collagen deposition (%) were measured using the ImageJ program. n=5 biologically independent mice / group were analyzed. Data are mean ± SEM. Scale bar 50 μm. (Figure 27B) Immunohistochemical analysis of β-catenin expression in kidneys of Wnt inhibitor-treated non-diabetic control mice and GRECKO mice. n=6 biologically independent mice / group combined from two independent experiments were analyzed. Representative images are shown; original magnification 300x. Statistical significance was calculated using one-way ANOVA with Tukey's multiple comparison post-hoc test. Significance; *p<0.05, **p<0.01, ***p<0.001. [Figure 28] Expression of Snail1, PPARα and HIF1α in Wnt inhibitor-treated non-diabetic and diabetic mice. Immunohistochemical analysis of Snail1, PPARα and HIF1α expression in Wnt inhibitor (Wnti)-treated and untreated control and GRECKO mice. Scale bar 50 μm. n=7 biologically independent mice / group combined from three independent experiments were analyzed. Representative images shown; original magnification 200x. [Figure 29] Figure 1 shows Snail1, PPARα, active p-catenin and HIF1α in isolated cells from Wnt inhibitor-treated diabetic mice. Western blot analysis of Snail1, PPARα, active p-catenin and HIF1α proteins in isolated endothelial cells. Representative blots are shown. Densitometry calculations were combined from three independent experiments. Statistical significance was calculated using one-way ANOVA with Tukey's multiple comparison post-hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. [Diagram 30]EndMT and EMT analysis in kidneys of Wnt inhibitor-treated mice. Immunofluorescence analysis of α-SMA / CD31 / DAPI and E-cadherin / α-SMA / DAPI in kidneys of Wnt inhibitor-treated non-diabetic and diabetic mice. Upper panel: α-SMA FITC-labeled, CD31 rhodamine-labeled, and DAPI blue; Lower panel: E-cadherin FITC-green labeled, α-SMA red rhodamine-labeled, and DAPI blue. Representative images shown; original magnification 400x. Scale bar 50 μm. n=6 biologically independent mice / groups combined from three independent experiments were analyzed. NC: non-diabetic control; DC: diabetic control; D: diabetic. [Diagram 31] Figures 31A-31B show fatty acid uptake and fatty acid oxidation in isolated endothelial cells. (Figure 31A) Fatty acid uptake was measured as [14C]palmitate incorporation. (Figure 31B) Fatty acid oxidation expressed by [14CO2] release. [14C]palmitate was used as substrate and CPM counts were normalized to protein. In both cases, isolated endothelial cells from n=5 biologically independent mice / group were analyzed in triplicates. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey multiple comparison post-hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. [Diagram 32] Gene expression of fatty acid transporter proteins in non-diabetic and diabetic mice is shown. Relative mRNA levels of Fatp1, Fatp4, and Cd36 were analyzed by qRT-PCR in endothelial cells isolated from kidneys of non-diabetic and diabetic control and GRECKO mice. Endothelial cells isolated from n=5 biologically independent mice / group were analyzed in triplicates. Data are mean ± SEM. One-way ANOVA with Tukey post-hoc test was used to analyze statistical significance. Significance: *p<0.05. [Figure 33-1]Figures 33A-33F show that endothelial GR is essential for the action of anti-dyslipidemic drugs in diabetic kidney disease. (Figure 33A) Masson's trichrome staining (MTS) and Sirius red staining in kidneys of control mice, diabetic mice, and diabetic mice treated with fenofibrate, etomoxir, C75, and simvastatin. Representative images (original magnification 300x) are shown. Relative fibrosis area (%) and relative collagen deposition (%) were measured using the ImageJ program. n=6 biologically independent mice / group combined from two experiments were analyzed. Scale bar 50 μm. (Figure 33B) Co-immunolabeling of GR / CD31 was analyzed by fluorescence microscopy. FITC-labeled GR, rhodamine-labeled CD31, and DAPI were used. Representative images are shown; original magnification 300x. n=6 biologically independent mice / group combined from two experiments were analyzed. [Figure 33-2] (Fig. 33C) PCR gene analysis of fibronectin and α-SMA in kidney. 18S was used as an internal control. n=6 biologically independent mice / group. (Fig. 33D) Blood glucose measurements in control mice, diabetic mice, diabetic mice treated with fenofibrate (Feno), etomoxir (Etomo), C75 and simvastatin (Sim). n=6 biologically independent mice / group. (Fig. 33E) Oxidation of radiolabeled [14C] palmitic acid and [14CO2] release were measured. CPM for each sample were counted. Endothelial cells isolated from six biologically independent mice were evaluated. Three independent experiments were analyzed. Data are mean ± SEM. (Fig. 33F) Immunohistochemical analysis of CPT1a and β-catenin in kidney of control mice, diabetic mice, diabetic mice treated with fenofibrate, C75, etomoxir and simvastatin. n=6 biologically independent mice / group. Representative images are shown; original magnification 400x. Scale bar 50 μm. Data are mean ± SEM. In all panels, statistical significance was calculated using one-way ANOVA with Tukey multiple comparison post-hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. Con: control, DM: diabetes, Feno: fenofibrate. [Diagram 34] Physiological characteristics of non-diabetic etomoxir-treated and C75-treated mice. Body weight, blood glucose, kidney / body weight ratio, and fatty acid oxidation expressed as [14CO2] are shown. [14C]palmitate was used as substrate and CPM counts were normalized to protein. n=6 biologically independent mice / group. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey multiple comparison post-hoc test. Significance: *p<0.05. [Diagram 35] Histological analysis of kidneys from non-diabetic etomoxir- and C75-treated mice. Masson's trichrome staining (upper panel) and Sirius red staining (lower panel) in etomoxir- and C75-treated non-diabetic control and GRECKO mice. Etomoxir (20 mg / kg) and C75 (15 mg / kg) were administered (IP) three times a week for 3 weeks to GRECKO and control littermates. Representative images shown; original magnification 400x. Fibrosis area (%) and relative collagen deposition (%) in n=6 biologically independent mice / group combined from two independent experiments. Scale bar 50 μm. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey's multiple comparison post-hoc test. Significance: **p<0.01. [Diagram 36] Figure 1 shows that GR overexpression suppresses fibrogenic markers in vitro. Western blots and corresponding quantification of protein levels of GR and collagen I (normalized to β-actin) and β-catenin and α-SMA (normalized to HSP90) in HUVECs exposed to either empty vector, GR overexpression vector, empty vector + TGFβ2, or GR overexpression vector + TGFβ2. Representative blots are shown. Combined data from three independent experiments were analyzed. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey's multiple comparison post-hoc test. Significance: ***p<0.001. [Figure 37]EC-specific inhibition of canonical Wnt resulted in significant amelioration of renal and cardiac fibrosis, providing support for the involvement of the Wnt canonical pathway.A novel mouse model that inhibits canonical Wnt signaling at the LRP5 / 6 receptor, the endothelial-specific LRP5 / 6 KO mouse model, was used to generate these data. [Figure 38] Figures 38A-38H show that pharmacological inhibition of Wnt signaling abolishes the fibrotic phenotype in db / db mice. (Figure 38A) Measurement of postprandial blood glucose (blood glucose) in db / db mice treated with Wnt inhibitors and untreated control db / db mice. LGK-974 (Wnt inhibitor) was administered by gavage at a dose of 5 mg / kg body weight. (Figure 38B) GTT analysis in db / db mice treated with Wnt inhibitors and untreated control db / db mice. n=6 were analyzed in each group. (Figure 38C) Area under the curve (AUC) in db / db mice treated with Wnt inhibitors and untreated control db / db mice. n=6 were analyzed in each group. (Figure 38D) Physiological parameters including body weight, epididymal fat, kidney weight, liver weight and heart weight in db / db mice treated with Wnt inhibitors and untreated control db / db mice. n=6 were analyzed in each group. (FIG. 38E) MTS and Sirius red staining analysis in Wnt inhibitor-treated and untreated control db / db mice in db / db mice. Representative photographs at 30× are shown. n=6 were analyzed in each group. Relative fibrosis area (RAF) and relative collagen deposition (RCD) were quantified using ImageJ software program. (FIG. 38F) MTS and Sirius red staining analysis of heart sections in Wnt inhibitor-treated and untreated control db / db mice in db / db mice. Representative photographs at 20× are shown. n=6 were analyzed in each group. (FIG. 38G) MTS of liver sections, (FIG. 38H) MTS of adipose tissue sections in Wnt inhibitor-treated and untreated control db / db mice in db / db mice. Representative photographs at 20× are shown. n=6 were analyzed in each group. Scale bar: 50 μm. All data represent mean±SEM. Unpaired t-test was used for statistical significance. *P≦0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0026] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a composition containing a "compound" includes a mixture of two or more compounds. It should also be noted that the term "or" is generally used in the sense of including "and / or" unless the context clearly dictates otherwise.

[0027] The term "antibody" refers to all isotypes of immunoglobulins (e.g., IgG, IgA, IgE, IgM, IgD and IgY), including the various monomeric, multimeric and chimeric forms, unless otherwise specified. In particular, the term "antibody" encompasses polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. Immunoglobulin molecules can be of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1 and IgA2) or subclass.

[0028] The term "antigen-binding fragment" refers to any proteinaceous structure that can exhibit binding affinity to a specific antigen. Antigen-binding fragments include those produced by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments are composed of a portion of an intact antibody that retains the antigen-binding specificity of the parent antibody molecule. For example, an antigen-binding fragment may contain at least one variable region (either heavy or light chain variable region) or one or more complementarity determining regions (CDRs) of an antibody known to bind to a specific antigen. Examples of suitable antigen-binding fragments include single chain molecules such as Fab, F(ab')2, Fc, Fabc, Fv molecules, scFv, and disulfide-linked Fv (sdFv), intrabodies, diabodies, minibodies, linear antibodies, single domain antibodies such as sdAbs (either VL or VH), camelid nanobodies (VHH domains), multispecific antibodies formed from antibody fragments, individual antibody light chains, individual antibody heavy chains, chimeric fusions of antibody chains or CDRs with other proteins, protein scaffolds, and the like. Antigen-binding fragments include, but are not limited to, monovalent fragments consisting of VL, VH, CL and CH1 domains, monovalent antibodies as described in WO2007059782 (incorporated herein by reference in their entirety), bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, Fd fragments consisting essentially of VH and CH1 domains, dAb fragments, or isolated CDRs. All antibody isotypes can be used to generate antigen-binding fragments. In addition, antigen-binding fragments can include non-antibody proteinaceous frameworks, such as protein scaffolds, into which polypeptide segments can be successfully incorporated in an orientation that confers affinity for a given antigen of interest. The phrase "antibody or antigen-binding fragment thereof" can be used to indicate that a given antigen-binding fragment incorporates one or more amino acid segments of the antibody referred to in the phrase.

[0029] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as the paratope. A single antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on the antigen and have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0030] The terms "patient", "individual", "subject", "mammal" and "animal" are used interchangeably herein and refer to mammals, including but not limited to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models (e.g., mice, rabbits, rats). Animals include all vertebrates, e.g., mammals and non-mammals, e.g., mice, sheep, dogs, cows, birds, ducks, geese, pigs, chickens, amphibians, and reptiles, etc. In a preferred embodiment, the subject is a human. In some embodiments, the subject is in need of prevention or treatment of dyslipidemia or related disorders or conditions.

[0031] The term "treat" or "treatment" of a condition, disorder or condition includes: (1) preventing, delaying, or reducing the likelihood of onset and / or appearance of at least one clinical or subclinical symptom of the condition, disorder or condition developing in a subject who may be afflicted with or susceptible to the condition, disorder or condition, but who has not yet experienced or displayed a clinical or subclinical symptom of the condition, disorder or condition; or (2) inhibiting the condition, disorder or condition, i.e., arresting, reducing, or delaying the onset of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (3) ameliorating the disease, i.e., causing regression of the condition, disorder or condition, or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated is statistically significant or at least perceptible to the patient or physician.

[0032] As used herein, the term "in need of treatment" refers to the judgment by a physician or other caregiver that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors within the physician's or caregiver's expertise.

[0033] The terms "therapeutically effective amount" and "effective amount" are used interchangeably herein and refer to the administration of an agent to a subject, either alone or as part of a pharmaceutical composition, and either in a single dose or as part of a series of doses, in an amount that, when administered to a subject, can produce any detectable positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition. A therapeutically effective amount can be ascertained by measuring the relevant physiological effect, and can be adjusted in conjunction with dosing regimens and diagnostic analyses of the subject's condition, and the like.

[0034] As used herein, the term "pharmaceutical acceptable" refers to molecular entities and other components of a composition that are physiologically tolerated and typically do not cause adverse reactions when administered to a mammal (e.g., a human). Preferably, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, more specifically, humans.

[0035] As used herein, the term "carrier" or "pharmaceutical acceptable carrier" refers to any clinically useful solvent, diluent, adjuvant, excipient, recipient, vehicle, and the like for use in preparing a mixture of a pharmaceutical composition.

[0036] The term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within an acceptable standard deviation, according to the practice of the art. Alternatively, "about" can mean within a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and even more preferably up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 2-fold, of the value. When a particular value is described in the present application and claims, unless otherwise stated, the term "about" is implicit and, in this context, means within an acceptable error range for the particular value.

[0037] As used in this specification and claims, the terms "comprising" (including any of its derivatives such as "comprising"), "having" (including any of its derivatives such as "having"), "including" (including any of its derivatives such as "comprising") or "containing" (and including any of its derivatives such as "containing") are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0038] In accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. See, inter alia, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 (herein referred to as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (Glover ed. 1985); Oligonucleotide Synthesis (Gait ed. 1984); Nucleic Acid Hybridization (Hames and Higgins eds. 1985); Transcription and Translation (Hames and Higgins eds. 1984); Animal Cell Culture (Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel et al. eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. See, e.g., 1994.

[0039] In one aspect of the disclosure, a method is provided for treating a condition or disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of IL-6 activity.

[0040] Conditions or diseases that may be treated by the methods of the present disclosure include, but are not limited to, organ fibrosis, nephropathy, dyslipidemia, hypertension, hyperlipidemia, hypercholesterolemia, cardiovascular disease, peripheral artery disease, atherosclerosis, coronary artery disease, coronary heart disease, and stroke.

[0041] In some embodiments, the organ fibrosis is renal fibrosis.

[0042] In some embodiments, the nephropathy is diabetic nephropathy.

[0043] In some embodiments, the method is effective for preventing or treating nephropathy. In some embodiments, the method is effective for reducing fibrosis in the kidney of a subject. In some embodiments, the method is effective for reducing collagen deposition in the kidney of a subject. In some embodiments, the method is effective for reducing collagen accumulation in the kidney of a subject. In some embodiments, the method is effective for reducing glomerulosclerosis in a subject.

[0044] The term "dyslipidemia" refers to abnormal levels of lipoproteins in plasma and includes both low and / or high levels of lipoproteins (e.g., high levels of LDL and / or VLDL, and low levels of HDL).

[0045] As used herein, the term "hypercholesterolemia" refers to a condition in which cholesterol levels are elevated above a desired level. In some embodiments, this means that serum cholesterol levels are elevated. In some embodiments, the desired level takes into account various "risk factors" known to those of skill in the art (and described or referenced herein). "Familial hypercholesterolemia" refers to hypercholesterolemia caused by a mutation in a gene located on chromosome 19.

[0046] The term "nephropathy" as used herein refers to disease, dysfunction or non-function of one or both kidneys. The term "diabetic nephropathy" as used herein includes both early and overt stages of diabetic nephropathy, whether diagnosed or not, although diabetic nephropathy is most typically diagnosed by a clinician or physician.

[0047] As used herein, the term "atherosclerosis" refers to a disease of arteries characterized by the narrowing of arteries due to the accumulation of plaque in arteries. The term "atherosclerosis-related disorder" refers to atherosclerotic cardiovascular disease (ASCVD) and other such chronic inflammatory diseases caused by cholesterol deposits. Atherosclerosis-related disorders include, but are not limited to, ASCVD; coronary heart disease, such as myocardial infarction, angina, and coronary artery stenosis; cerebrovascular disease, such as transient ischemic attack, ischemic stroke, and carotid artery stenosis; peripheral artery disease, such as claudication; aortic atherosclerotic disease, such as abdominal aortic aneurysm and descending thoracic aortic aneurysm; hypertension; peripheral vascular disease; coronary artery disease; aortic aneurysm; carotid artery disease; coronary atherosclerosis; heart attack; acute coronary syndrome, and stroke.

[0048] As used herein, the term "coronary heart disease" refers to the narrowing of the small blood vessels that supply blood and oxygen to the heart, often as a result of atherosclerosis.

[0049] In various embodiments of the methods described herein, the subject is a human.

[0050] In some embodiments, the inhibitor of IL-6 activity is an antibody or antigen-binding fragment.

[0051] In some embodiments, the inhibitor is a neutralizing antibody of IL-6 or an antigen-binding fragment thereof. In some embodiments, the inhibitor is sirukumab, siltuximab, or olokizumab, or an antigen-binding fragment thereof.

[0052] In some embodiments, the inhibitor is an antibody or antigen-binding fragment thereof that targets the IL-6 receptor. In some embodiments, the inhibitor is tocilizumab or sarilumab, or an antigen-binding fragment thereof.

[0053] In some embodiments, the antibodies or antigen-binding fragments described herein are, for example, about 1×10 -8 K under M D and specifically binds to IL-6 or the IL-6 receptor with high affinity, e.g., K D is approximately 1 to 9.9 (or any range or value therebetween, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, etc.) x 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, 10 -15M, or any range or value therebetween, as determined, for example, by biolayer interferometry assays, surface plasmon resonance, or Kinexa methods, as performed by one of skill in the art. One exemplary affinity is 1×10 -10 Another exemplary affinity is about 3.8x10 -11 Equal to M.

[0054] Methods for testing antibodies for the ability to bind to a target peptide or any portion thereof are known in the art and include any antibody-antigen binding assay, such as, for example, biolayer interferometry assays, radioimmunoassays (RIA), Western blots, enzyme-linked immunosorbent assays (ELISAs), immunoprecipitations, and competitive inhibition assays. In some embodiments, the K D Values ​​are determined using a Biolayer Interferometry assay.

[0055] In some embodiments, the antibodies or antigen-binding fragments described herein are human antibodies, monoclonal antibodies, humanized antibodies, single chain antibodies, Fab, Fab', F(ab')2, Fv, or scFv.

[0056] Antibodies or antigen-binding fragments thereof that compete with the antibodies or antigen-binding fragments described herein for binding to IL-6 or IL-6 receptor may also be used in the methods of the present disclosure. The term "compete" or "cross-compete" as used herein means that an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. The term also includes competition between two antibodies in both orientations (a first antibody binds to and blocks the binding of a second antibody, and vice versa). In some embodiments, a competing antibody and an antibody described herein may bind to the same epitope. Alternatively, the competing antibody and an antibody described herein bind to different but overlapping epitopes, such that the binding of one antibody inhibits or blocks the binding of the second antibody, for example, through steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, for example, by label-free biolayer interferometry assays in real time. Cross-competition between two antibodies can be expressed as the binding of the second antibody less than the background signal due to self-self binding (where the first and second antibodies are the same antibody). Cross-competition between two antibodies can be expressed, for example, as the % binding of the second antibody less than the baseline self-self background binding (where the first and second antibodies are the same antibody).

[0057] Antibodies or antigen-binding fragments that bind to the same epitope as the antibodies or antigen-binding fragments described herein can also be used in the methods of the disclosure.

[0058] The antibody or antigen-binding fragment described herein may be any of various antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. In some embodiments, the antibody isotype is IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody isotype is IgA1 or IgA2. The specificity of an antibody or its antigen-binding fragment is determined primarily by the amino acid sequence and arrangement of the CDRs. Thus, the CDRs of one isotype can be transferred to another isotype without changing the antigen specificity. Alternatively, there are established techniques for switching hybridomas from producing one antibody isotype to producing another antibody isotype without changing the antigen specificity (isotype switching). Thus, such antibody isotypes are within the scope of the described antibody or antigen-binding fragment.

[0059] In some embodiments, the inhibitor of IL-6 activity results in a blockade of trans-signaling. In one embodiment, the inhibitor is olamxept (FE999301), or a fragment or variant thereof.

[0060] In some embodiments, the inhibitor of IL-6 activity is an inhibitor of intracellular signaling. In some embodiments, the inhibitor is tofacitinib or CpG-stat3 siRNA.

[0061] The polypeptide compounds (such as antibodies or antigen-binding fragments) described herein include variants having single or multiple amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described polypeptide compounds.

[0062] These variants may include: (i) variants in which one or more amino acid residues are replaced with conservative or non-conservative amino acids, (ii) variants in which one or more amino acids are added to or deleted from the polypeptide, (iii) variants in which one or more amino acids include a substituent, and (iv) variants in which the antibody or antigen-binding fragment described is fused or conjugated to another peptide or polypeptide (e.g., fusion partner, protein tag) or to other chemical moieties that can confer useful properties to the antibody or antigen-binding fragment (e.g., antibody epitopes, polyhistidine sequences, biotin moieties, etc.). The polypeptide compounds described herein may include variants in which amino acid residues from one species are replaced with corresponding residues in another species, either at conserved or non-conserved positions. In other embodiments, amino acid residues at non-conserved positions are replaced with conservative or non-conserved residues. Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, such as, for example, their hydrophobicity, hydrophilicity, charge, size, etc.

[0063] Amino acid substitutions can be conservative, meaning that the substituted amino acid has similar chemical properties as the original amino acid. Those skilled in the art will understand which amino acids share similar chemical properties. For example, the following amino acid groups share similar chemical properties such as size, charge and polarity: Group I (Ala, Ser, Thr, Pro, Gly); Group II (Asp, Asn, Glu, Gln); Group III (His, Arg, Lys); Group IV (Met, Leu, Ile, Val, Cys); Group V (Phe, Thy, Trp).

[0064] Thus, embodiments of the polypeptide compounds can include variants having about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the described polypeptide compounds.

[0065] In some embodiments of the method provided herein, the method further comprises administering to the subject one or more additional drugs effective for treating the intended condition or disease.Such drugs may include, but are not limited to, cholesterol-lowering drugs (e.g., statins, fibrates, inhibitors of proprotein convertase subtilisin / kexin type 9), antihypertensive therapeutic drugs (e.g., angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs)), anti-inflammatory drugs, antithrombotic drugs, anticoagulants, renin-angiotensin-aldosterone system inhibitors (RAAS inhibitors), β-adrenergic blockers, calcium channel blockers, hypoglycemic drugs (e.g., metformin, insulin, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide 1 (GLP-1), sodium / glucose cotransporter 2 (SGLT2) inhibitors), and / or other treatment modalities of non-pharmacological nature. When combination therapy is used, the IL-6 inhibitor and one additional agent may be in the form of a single composition or in the form of multiple compositions, and the treatment modalities may be administered simultaneously, sequentially, or through some other regimen. Combination therapy may have additive or synergistic effects.

[0066] Other suitable agents that may be used in combination with the IL-6 inhibitors described herein include, but are not limited to, the Wnt inhibitors described in U.S. Pat. Nos. 11,369,609 and 9,045,416, which are incorporated herein by reference in their entireties.

[0067] For example, a Wnt inhibitor used in combination with an IL-6 inhibitor described herein may include a compound having a structure according to formula (I), or a pharma- ceutically acceptable salt thereof: [ka] [In the formula, X1 and X2 are selected from N and CR; one of X3, X4, X5 and X6 is N, and the others are selected from N and CR; X7, X8, X9 and X 10 one of is N and the others are selected from N and CR; X 11 , X 12 , X 13 and X 14 one of is N and the others are selected from N and CR; R is independently at each occurrence selected from hydrogen, halo, cyano, methyl, difluoromethyl, and trifluoromethyl.

[0068] In some embodiments of the above compounds, one of X3, X4, X5, and X6 is N, and the others are CR. In some embodiments, X7, X8, X9, and X 10 In some embodiments, one of X is N and the other is CR. 11 , X 12 , X 13 and X 14 Two of are N and the other is CR. In some embodiments, X1 is CR, R is methyl, and / or X5 is CR and R is methyl. In some embodiments, one or more of X2 is CH, X4 is CH, X6 is CH, X8 is CH, X9 is CH, and X 10 is CH and X 12 is CH and X 13 is CH.

[0069] In a specific embodiment, the compound is a compound having the structure of Formula (I), wherein: X1 is CR, where R is methyl; X2 is CH; X3 is N; X5 is CR, where R is methyl; X4 and X6 are each CH; X7 is N; X8, X9 and X 10 are CH, respectively; X 11 and X 14 are N and X 12 and X 13 are CH, respectively.

[0070] In certain embodiments, the Wnt inhibitor used in combination with the IL-6 inhibitors described herein may include the compound LGK974, having the following structure, or a pharma- ceutically acceptable salt thereof: [ka]

[0071] Without wishing to be bound by theory, LGK974 can inhibit PORCN, which is understood to be necessary for palmitoylation of Wnt ligand.LGK974 can inhibit one or more of the following Wnt ligands: Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5B, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16.LGK974 can also inhibit the phosphorylation of LRP5 and LPR6.

[0072] Chemical structures in this specification are drawn according to conventional standards known in the art.Therefore, if an atom such as a carbon atom is drawn and is deemed to have insufficient valence, its valence is assumed to be satisfied by a hydrogen atom, even if the hydrogen atom is not necessarily explicitly drawn.Hydrogen atoms should be assumed to be part of the compound.

[0073] As will be understood by those skilled in the art, description of a compound is intended to include the salts, solvates, oxides, and inclusion complexes of the compound, as well as any stereoisomeric form, or mixtures of any such forms of the compound in any ratio. The compounds described herein include, but are not limited to, their optical isomers, racemates, and other mixtures thereof. In these circumstances, single enantiomers or diastereomers, i.e., optically active forms, can be obtained by asymmetric synthesis or by resolution of the racemates. Resolution of racemates can be achieved by conventional methods, such as, for example, crystallization in the presence of a resolving agent, or chromatography, for example, using a chiral high pressure liquid chromatography (HPLC) column. Furthermore, such compounds include Z- and E-forms (or cis- and trans-forms) of compounds having carbon-carbon double bonds. In the case where the compounds described herein exist in various tautomeric forms, the term "compound" is intended to include all tautomeric forms of the compound. Such compounds include crystalline forms, including polymorphs and clathrates. Similarly, the term "salt" is intended to include all tautomeric and crystalline forms of the compound.

[0074] Thus, according to some embodiments of the present disclosure, the compounds described herein are provided as salt forms, including in the context of pharmaceutical compositions and methods of treatment. A "pharmaceutical acceptable salt" of a compound refers to a pharmaceutically acceptable salt of a compound. Desirable are salts of compounds that retain or improve the biological effectiveness and properties of the free acids and bases of the parent compounds as defined herein, or salts of compounds that utilize inherently basic, acidic or charged functional groups on the molecule, and are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts are described, for example, in Berge et al., "Pharmaceutical Salts", J. Pharm. Sci. 66, 1-19 (1977).Non-limiting examples of such salts include: (1) those formed by the addition of inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, carbonate forming agents, to basic or positively charged functional groups; or those formed with acetic acid, propionic acid, lactic acid, oxalic acid, glycolic acid, pivalic acid, t-butyl acetate, beta-hydroxybutyric acid, valeric acid, hexanoic acid, cyclopentanepropionic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, or the like. , 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, cyclohexylaminosulfonic acid, benzenesulfonic acid, sulfanilic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 3-phenylpropionic acid, laurylsulfonic acid, lauryl sulfate, oleic acid, palmitic acid, stearic acid, lauric acid (2) acid addition salts formed with organic acids such as embonic acid (pamoic acid), palmitic acid, pantothenic acid, lactobionic acid, alginic acid, galactaric acid (mucic acid), galacturonic acid, gluconic acid, glucoheptonic acid, glutamic acid, naphthoic acid, hydroxynaphthoic acid, salicylic acid, ascorbic acid, stearic acid, muconic acid, and the like; (3) acid addition salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion (lithium, sodium, potassium, etc.), an alkaline earth ion (magnesium, calcium, barium, etc.), or another metal ion such as aluminum, zinc, iron, and the like; or base addition salts formed when the acidic proton present in the parent compound is coordinated with an organic base such as ammonia, ethylamine, diethylamine, ethylenediamine, N,N'-dibenzylethylenediamine, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, piperazine, chloroprocaine, procaine, choline, lysine, and the like.

[0075] Pharmaceutically acceptable salts can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally, such salts are prepared by reacting a compound in its free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or in a mixture of the two. Salts can be prepared during the final isolation or purification of a compound, or in situ by separately reacting a compound in its free acid or base form with the desired corresponding base or acid and isolating the salt thus formed. The term "pharmaceutical acceptable salt" includes zwitterionic compounds that contain a cationic group covalently bonded to an anionic group. It is understood that all acid, salt, base, and other ionic and non-ionic forms of the compounds described herein are intended to be encompassed. For example, when a compound is referred to herein as an acid, the salt form of the compound is also encompassed. Similarly, when a compound is referred to as a salt, the acid and / or base form is also encompassed.

[0076] The pharmaceutical composition of the present disclosure may comprise a compound described herein and a pharma- ceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers may comprise, for example, physiologically acceptable compounds that act to stabilize, or increase or decrease the absorption or clearance rate of the pharmaceutical composition. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, compositions that reduce the clearance or hydrolysis of low molecular weight proteins, glycopeptides, or excipients or other stabilizers and / or buffers. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives that are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known, and include, for example, phenol and ascorbic acid. Surfactants, including liposomal carriers, may also be used to stabilize, or increase or decrease the absorption of the pharmaceutical composition. Pharmaceutically acceptable carriers and formulations are known to those of skill in the art and are described extensively in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. ("Remington's"). One of skill in the art will understand that the choice of a pharma- ceutically acceptable carrier, including a physiologically acceptable compound, will depend, for example, on the route of administration of the composition and its particular physiochemical characteristics.

[0077] The composition may be administered by any suitable means, for example, orally in the form of pills, tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular, intraperitoneal or intrasternal injection, or by using infusion techniques (e.g., as a sterile injectable aqueous or non-aqueous solution or suspension); intranasally, such as by inhalation spray, aerosol, mist, or nebulizer; topically, such as in the form of a cream, ointment, salve, powder, or gel; transdermally, such as in the form of a patch; transmucosally; rectally, such as in the form of a suppository. The composition may be administered in a form suitable for immediate or sustained release. Immediate or sustained release may be achieved by the use of suitable pharmaceutical compositions, or, particularly in the case of sustained release, by the use of devices such as subcutaneous implants or osmotic pumps.

[0078] In various embodiments, pharmaceutical compositions are formulated for oral administration.Forms suitable for oral administration include, but are not limited to, tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, liquids, microbeads or elixirs.The pharmaceutical compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more substances, such as, for example, sweeteners, flavorings, colorings and preservatives, to provide pharmaceutically acceptable preparations.Tablets, capsules and the like usually contain the active ingredient in a mixture with non-toxic pharmaceutically acceptable carriers or excipients suitable for the manufacture of tablets. These carriers or excipients may be, for example, diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc.

[0079] Tablets, capsules, etc. suitable for oral administration may be uncoated or may be coated using known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action. For example, time-delay materials such as glyceryl monostearate or glyceryl distearate may be used. They may be coated by techniques known in the art to form osmotic therapeutic tablets for controlled release. Additional agents include biodegradable or biocompatible particles or polymeric materials, such as polyesters, polyamic acids, hydrogels, polyvinylpyrrolidones, polyanhydrides, polyglycolic acids, ethylene vinyl acetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide / glycolide copolymers, polylactide / glycolide copolymers, or ethylene vinyl acetate copolymers, to control the delivery of the administered composition. For example, oral agents may be encapsulated in microcapsules prepared by coacervation techniques or interfacial polymerization using hydroxymethylcellulose or gelatin-microcapsules or poly(methyl methacrylate) microcapsules, respectively, or may be encapsulated in colloidal drug delivery systems. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, microbeads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. Methods for the preparation of the above-mentioned formulations will be apparent to those skilled in the art.

[0080] Formulations for oral use may also be provided as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, kaolin or microcrystalline cellulose, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil. Aqueous suspensions contain the active material mixed with excipients suitable for their manufacture. Such excipients include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, such as naturally occurring phospholipids (e.g., lecithin), or condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearates), or condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides (e.g., polyethylene sorbitan monooleate), and the like. Aqueous suspensions may also contain one or more preservatives.

[0081] Other suitable preparations for oral use include oily suspensions.Oily suspensions can be prepared by suspending active ingredient in vegetable oil such as peanut oil, olive oil, sesame oil or coconut oil, or mineral oil such as liquid paraffin.Oily suspensions can contain thickening agent such as beeswax, solid paraffin or cetyl alcohol.Sweetener and flavoring agent as mentioned above can be added to provide the oral preparations that are easy to eat.

[0082] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are known in the art.

[0083] The pharmaceutical composition of the present disclosure may be in the form of oil-in-water emulsion.The oil phase may be vegetable oil, such as olive oil or peanut oil, or mineral oil, such as liquid paraffin, or mixtures thereof.Suitable emulsifiers are natural gums, such as gum acacia or gum tragacanth; natural phospholipids, such as soybean lecithin, and esters or partial esters derived from fatty acids; anhydrous hexitol, such as sorbitan monooleate; and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.

[0084] The pharmaceutical compositions of the present disclosure may be manufactured into useful dosage units for administration by a variety of routes, such as topical, oral, subcutaneous, intravenous, and intranasal administration, among others.

[0085] The pharmaceutical composition of the present disclosure can also contain other bioactive substances in combination with the compound of the present disclosure.These additional bioactive substances can also be formulated as separate compositions and can be administered simultaneously or sequentially with the compound of the present disclosure.Non-limiting examples of useful bioactive substances include statins, niacin, bile acid resins, fibric acid derivatives, cholesterol absorption inhibitors, and other lipid-lowering drugs.

[0086] The optimal therapeutically effective amount of a compound or composition of the present disclosure may be determined empirically, taking into account the exact mode of administration, the form in which the drug is administered, the indication for which it is being administered, the subject involved (e.g., weight, health, age, sex, etc.), and the preferences and experience of the attending physician or veterinarian.

[0087] Following methodologies well established in the art, the effective dosage and toxicity of compounds and compositions of the present disclosure that have shown good results in in vitro tests can be determined in studies using small animal models (e.g., mice, rats) in which the drugs are found to be therapeutically effective and can be administered by the same routes proposed for human clinical trials.

[0088] For any pharmaceutical composition used in the method of the present disclosure, the dose-response curve obtained from an animal system can be used to determine the test dose for administration to humans.In determining the safety of each composition, the dose and frequency of administration should meet or exceed the dose and frequency expected for use in clinical trials.

[0089] As disclosed herein, the dosage of the disclosed compound or composition is determined to ensure that the dosage administered continuously or intermittently does not exceed the amount determined after considering the results in test animals and the individual condition of the patient.The specific dosage naturally varies according to the condition of the patient or subject animal, such as administration procedure, age, body weight, sex, sensitivity, feed, administration period, concomitant drugs, disease severity, etc. (finally determined according to the doctor's judgment and the circumstances of each patient).

[0090] Toxicity and therapeutic efficacy of the compositions of the present disclosure can be determined in experimental animals by standard pharmaceutical procedures, e.g., LD 50 (a dose lethal to 50% of the population) and ED 50 The dose ratio between therapeutic and toxic effects is the therapeutic index, and the ED 50 / LD 50 It can be expressed as a ratio.

[0091] The compounds of the present disclosure can be formulated for parenteral, oral, topical, transdermal, transmucosal, intranasal, buccal, or any other standard route of administration. Parenteral administration includes intravenous (iv), subcutaneous (sc), intraperitoneal (ip), intramuscular (im), subdermal (sd), intradermal (id), intra-articular, intrasynovial, intra-arterial, intraventricular, intrathecal, intrasternal, intrahepatic, intralesional, or intracranial administration, direct injection, e.g., bolus injection, continuous infusion, or via gene gun. The preferred route of administration according to the present disclosure depends mainly on the indication to be treated, and includes topical, oral, subcutaneous, intravenous, and intranasal administration, among others.

[0092] Preparations for injection can be provided in unit dosage form, for example in ampoules, or in multi-dose containers with the addition of preservatives. The compositions can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulating agents, such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient can be in powder form for reconstitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. Formulations suitable for parenteral administration can contain substances that increase viscosity, such as, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. Optionally, the formulation can contain stabilizers. In addition, the compounds of the present disclosure can be administered encapsulated in liposomes. Depending on their solubility, the compounds can be present in both the aqueous and lipid phases, or in what is commonly referred to as liposomal suspensions. The hydrophobic layer typically, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, some ionic surfactants such as diacetyl phosphate, stearylamine or phosphatidic acid, and / or other substances that are hydrophobic.

[0093] In certain embodiments, the compound and / or composition of the present disclosure is formulated for oral administration.For oral administration, the formulation of the present disclosure can be in the form of tablet or capsule prepared by conventional means using pharmaceutically acceptable excipients such as binder (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); filler (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricant (e.g., magnesium stearate, talc or silica); disintegrant (e.g., potato starch or sodium starch glycolate); or wetting agent (e.g., sodium lauryl sulfate).Tablet can be coated by methods well known in the art. The compositions of the present disclosure can also be incorporated into microspheres or microcapsules, for example, made from polyglycolic acid / lactic acid (PGLA) (see U.S. Patent No. 5,814,344; U.S. Patent No. 5,100,669 and U.S. Patent No. 4,849,222; International Publication No. WO95 / 11010 and WO93 / 07861).The liquid preparations for oral administration can be in the form of, for example, solution, syrup, emulsion or suspension, or they can be provided as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means using pharma- ceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl p-hydroxybenzoate, or sorbic acid). Preparations can contain buffer salts, flavoring agents, coloring agents, and sweetening agents as necessary. Preparations for oral administration can be suitably formulated to give controlled release of the active compound.

[0094] For administration by inhalation, the therapeutic agent according to the present disclosure can be conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example of gelatin, for use in an inhaler or insufflator can be formulated to contain a powder mix of the compound and a suitable powder base, for example, lactose or starch.

[0095] In addition to the formulation described above, composition can also be formulated as depot preparation.Such long-acting preparation can be administered by implantation (for example, subcutaneous or intramuscular) or intramuscular injection.Thus, for example, compound can be formulated with suitable polymer or hydrophobic material (for example, as emulsion in acceptable oil) or with ion exchange resin, or as poorly soluble derivative, for example as poorly soluble salt. EXAMPLES

[0096] The following examples are provided to further illustrate some of the embodiments disclosed herein. The examples are intended to illustrate, but not limit, the disclosed embodiments.

[0097] Example 1. Endothelial GR deficiency leads to a fibrogenic phenotype in the kidneys of diabetic mice Renal fibrosis phenotype depends on mouse strain specificity 30 Therefore, streptozotocin (STZ)-induced diabetic CD-1 mice are an established mouse model for studying diabetic kidney disease. 29-31 Although STZ-induced diabetic CD-1 mice and diabetic C57BL / 6 mice have similar blood glucose levels, kidneys from diabetic CD-1 mice have been shown to have a higher rate of EndMT and more severe fibrosis compared to kidneys from diabetic C57BL / 6 mice.29、32 Therefore, diabetic CD-1 mice are considered to be a pro-fibrotic strain, whereas diabetic C57B / L6 mice are considered to be a hypofibrotic strain. 32、33 .

[0098] As assessed by immunofluorescence staining, kidneys from diabetic CD-1 mice showed significant suppression of GR compared to those from diabetic C57BL / 6 mice (Figure 1A). Furthermore, ECs isolated from kidneys of diabetic CD-1 mice showed dramatic suppression of both protein and mRNA levels of GR compared to diabetic C57BL / 6 mice and nondiabetic controls of both genotypes (Figure 1B). The effect of corticosterone deficiency on the progression of renal fibrosis was evaluated by following bilateral adrenalectomy in both strains of mice. The efficacy of the surgical procedure was verified by the substantial suppression of corticosterone levels in all mice studied (Figure 9A); adrenalectomy did not affect blood glucose (blood glucose levels) (Figure 9B). Adrenalectomy did not cause significant differences in the extent of fibrosis or collagen expression levels (Figure 9C), suggesting that the global suppression of corticosterone did not affect the observed fibrotic phenotype.

[0099] Example 2. Loss of ECGR exacerbates renal fibrosis Endothelial GR knockout (GR ECKO ) To verify efficient removal of GR from ECs in mouse kidneys, we performed Western blot and qPCR for GR in isolated renal endothelial cells. Flow cytometry analysis demonstrated that isolation of renal ECs was achieved with a purity of approximately 95% (Figure 10). As shown in Figures 11A-11B, mRNA and protein levels were significantly higher than those of GR, as expected. ECKO Diabetes mellitus was significantly reduced in ECs from 8-week-old GR mice. ECKO (GR fl / fl ;Tie1 Cre+) and Cre- littermate controls (GR fl / fl ) and GR fl / fl ;Tie1 Cre+ / Apoe - / -(DKO) mice and their littermates (GR fl / fl ;Apoe - / - ) were injected with a low dose of STZ (50 mg / kg / day IP) for 5 consecutive days (Figure 2A). Animals were monitored for 4 months after STZ treatment before sacrifice. At the time of sacrifice, nondiabetic and diabetic GRs were ECKO and DKO mice and their littermate controls showed no significant changes in body weight, blood glucose, heart weight, liver weight, triglycerides, or cholesterol; however, diabetic GR and DKO mice and their littermate controls showed no significant changes in body weight, blood glucose, heart weight, liver weight, triglycerides, or cholesterol; ECKO and diabetic DKOs had relatively higher kidney weights and albumin / creatinine ratios when compared to their corresponding diabetic controls (Figures 2B-2I, Figure 12). The systolic blood pressure of diabetic mice of all genotypes was approximately 20 mmHg lower than the corresponding nondiabetic controls, possibly due to the massive polyuria induced by diabetes, but was otherwise not significantly different (Figure 12). Diabetic DKOs had significantly higher kidney weights and albumin / creatinine ratios when compared to their corresponding diabetic controls (Figure 2B-2I, Figure 12). ECKO Diabetic GRs had significantly higher kidney weights and albumin / creatinine ratios compared with control. Renal fibrosis was assessed by histological analysis of kidney sections from all genotypes. ECKO Mice exhibited higher relative fibrosis area, higher relative collagen deposition, and more severe glomerulosclerosis at 4 months compared to diabetic littermate controls (Figure 2J, Figure S13A). Diabetic DKO mice exhibited higher relative fibrosis area, higher relative collagen deposition, and more severe glomerulosclerosis compared to diabetic littermate controls (Figure 2J, Figure S13A). - / - Control and diabetic GR ECKO The diabetic GR showed a significant increase in relative fibrosis area and relative collagen deposition compared to the control (Figure 2J). ECKO Mice showed higher expression of vimentin, snail1 and HIF1α and lower expression of PPARα at 4 months compared to diabetic littermate controls (Figure 13B; Figure 14). - / - A similar pattern was observed in diabetic DKOs when compared to controls (FIG. 13B; FIG. 14). Immunofluorescence data showed that GR ECKO showed higher collagen I and fibronectin deposition in the kidneys of diabetic animals, with the highest deposition observed in DKO mice (Figure 15).

[0100] To test the role of endothelial GR in nondiabetic fibrosis, 8-week-old GR ECKO Unilateral ureteral obstruction (UUO) was performed in control and control littermates (Figure 16A). ECKO There was no significant difference in renal fibrosis in the contralateral kidney of mice. ECKO The UUO kidneys from mice showed a larger relative fibrosis area and more collagen deposition compared to the UUO kidneys of littermate controls ( FIG. 16B ). Immunofluorescence staining showed that the GR kidneys had a larger relative fibrosis area and more collagen deposition compared to the UUO kidneys of control littermates. ECKO The UUO kidneys of C57BL / 6 mice showed greater deposition of collagen I, αSMA, and fibronectin (Figure 16C). Administration of the GR agonist dexamethasone abolished the renal fibrotic phenotype by day 10 in C57BL / 6 mice subjected to UUO (Figure 17A). Dexamethasone treatment slightly suppressed renal fibrosis at day 15 in the contralateral kidneys of C57BL / 6 mice but did not cause any significant changes in nondiabetic control CD-1 mice (Figures 17A-17B).

[0101] Example 3. Endothelial GR loss reprograms cytokine and chemokine homeostasis Inflammation is a key factor during the fibroblast activation process in the kidney of diabetic mice 34、35 Disruption of cytokine and chemokine homeostasis contributes to the development of diabetic kidney disease 36、38 To investigate whether homeostasis is disturbed in this model, we performed cytokine analysis in the plasma of diabetic mice with severe fibrosis (diabetic CD-1) and in the plasma of diabetic mice with less severe fibrosis (diabetic C57BL / 6). Diabetic CD-1 mice showed higher plasma IL-1β, IL-6, IL-10, IL-17, G-CSF, IFN-γ, TNF-α, MCP-1, CCL3 and CCL4 levels, whereas CCL5 levels were suppressed compared to those in diabetic C57BL / 6 mice (Figure 18). Diabetic and non-diabetic GR ECKO Mice and littermate controls and diabetic and non-diabetic DKO and Apoe - / -The same cytokines were also analyzed in plasma from controls. Diabetic GRs were significantly higher in the plasma from diabetic control littermates than in the plasma from the respective diabetic control littermates. ECKO A similar pattern was observed in both genotypes in that IL-1β, IL-6, IL-10, eotaxin, G-CSF and CCL4 were significantly higher, whereas CCL5 was significantly lower, in the plasma of diabetic GR and DKO mice (Figure 3A; Figure S1A). Similarly, mRNA gene expression analysis demonstrated that diabetic GR and DKO mice had significantly higher levels of CCL5 than diabetic GR and DKO mice (Figure S1B). ECKO We demonstrated that levels of IL-1β, IL-6, IL-10, IL-17, eotaxin, and CCL4, whereas CCL5, were significantly upregulated in kidneys of diabetic DKO mice compared with diabetic kidneys of their respective control littermates (Figure 19B), indicating enhanced EC inflammation in mice lacking endothelial GR.

[0102] Stimulation of isolated renal endothelial cells from CD-1 mice with recombinant IL-1β, IL-6, TNFα and TGFβ caused a significant increase in the expression of mesenchymal markers vimentin and αSMA (Figures 20A-20B); neutralization of IL-6 using IL-6 neutralizing antibody could suppress the expression of vimentin in TGFβ2-treated and untreated cells (Figure 20C). To further investigate the role of IL-6 in diabetes-associated renal fibrosis, we injected (IP) a neutralizing antibody of IL-6 into non-diabetic and diabetic CD-1 mice. In non-diabetic mice, neutralization of IL-6 did not result in any significant difference; however, in diabetic mice, neutralization of IL-6 led to a significant reduction in fibrosis and collagen deposition (Figure 20D). Furthermore, the diabetic GR ECKO Cells isolated from diabetic and DKO mice had higher IL-6 levels in the culture medium compared to cells isolated from their respective diabetic control littermates (Figure 3B). ECKO and DKO mice, completely reversed the fibrotic phenotype in diabetic animals compared to mice injected with the respective IgG controls (FIG. 3C). No significant effect was observed in non-diabetic mice (FIG. 21).

[0103] Example 4. Canonical Wnt signaling is a new drug target for the action of endothelial GR Recently reported regulation of Wnt signaling by endothelial GR 27 and the recognized role of Wnt signaling in renal fibrosis 39 Based on this, the present inventors have ECKO We assessed the mRNA expression of Wnt-dependent genes and fibrogenic markers in ECs isolated from the kidneys of diabetic GR and diabetic DKO mice and their diabetic littermate controls. ECKO and diabetic DKO kidneys were upregulated compared to their respective controls. However, kidneys of diabetic DKO mice showed the highest expression of both Wnt-dependent genes, such as axin2 and tcf, and fibrogenic markers, such as αSMA and fibronectin (Figure 14A). Diabetic GR was upregulated when compared to diabetic controls. ECKO In ECs isolated from the kidneys of diabetic GRKO mice, HIF1α and Snaill1 expression was upregulated, whereas PPARα expression was downregulated (Figure 22A). In ECs isolated from cultured glomeruli, expression of Wnt-dependent genes axin2 and tcf, as well as fibronectin, was upregulated in diabetic GRKO mice compared with littermate control mice and nondiabetic mice. ECKO The protein expression of β-catenin was also upregulated in diabetic GR. ECKO and diabetic DKOs compared to their respective controls (FIG. 22C). Western blotting showed that diabetic GR was significantly higher in diabetic DKOs compared to diabetic control littermates. ECKO We showed higher protein expression of vimentin, HIFlα, snail1, and active β-catenin, as well as reduced levels of PPARα and CPT1a in ECs isolated from diabetic DKO mice (Figure 4A). ECs isolated from diabetic DKO mice were significantly higher in ECs than their littermate Apoe - / - and diabetes GR ECKOCompared with mice, they showed higher fibronectin, αSMA, and active β-catenin levels ( Fig. 4B ).

[0104] A similar pattern was observed using immunofluorescence co-staining, and diabetic GR ECKO There was a higher level of αSMA / CD31 and TGFβR1 / CD31 co-staining in the kidneys of diabetic GR and DKO mice compared to their respective controls, suggesting EndMT (Figure 4C). ECKO There was no difference in endothelial cell density of ECs from kidneys of diabetic GR and diabetic DKO; however, endothelial cell permeability was greater in diabetic GR and DKO kidneys, as demonstrated by FITC-dextran staining. ECKO and in kidneys and isolated cultured ECs from diabetic DKOs compared with their respective control littermates (Figures 23A-23C).

[0105] Example 5. Inhibition of canonical Wnt signaling ameliorates renal fibrosis As assessed by immunohistochemical staining, kidneys from diabetic CD-1 mice showed significantly higher expression of β-catenin, a marker of canonical Wnt signaling, compared with those from diabetic C57BL / 6 mice (Figure 24A). Kidneys from diabetic CD-1 mice also showed dramatically higher mRNA expression of Wnt-dependent genes and fibronectin compared with those from diabetic C57BL / 6 mice and nondiabetic controls of both genotypes (Figure 24B). To determine whether inhibition of the Wnt signaling pathway could ameliorate the observed fibrosis, we used LGK974, a small molecule inhibitor of all secreted Wnts, to treat fibrosis observed in diabetic CD-1 mice. 40was used. Figure 24C-D shows a schematic diagram illustrating the experimental protocol for LGK974 treatment in diabetic CD-1 and UUO mice. LGK974 significantly reduced ECM deposition, relative fibrotic area, collagen accumulation and glomerular sclerosis in both models used (Figure 24E-24F). Wnt inhibition also substantially restored endothelial GR and suppressed the levels of β-catenin in diabetic and UUO mice (Figure 24G-24J). In diabetic CD-1 mice, LGK974 also significantly suppressed the increase in the levels of IL-1β, IL-6, IL-10, G-CSF, TNFα, MCP-1, and CCL4, while increasing the levels of CCL5 (Figure 25).

[0106] Example 6. Wnt inhibitors inhibit diabetes GR ECKO Partially suppresses fibrogenic phenotype in kidney of Wnt inhibition reduces diabetes GR ECKO To determine whether LGK974 could attenuate the renal fibrosis observed in mice, a cohort of animals was treated with LGK974. At 8 weeks of age, control and GR mice were ECKO Mice were injected with STZ at 50 mg / kg for 5 consecutive days. Sixteen weeks after injection, LGK974 was administered by gavage for 8 weeks (Figure 5A). At the time of sacrifice, there were no significant differences in body weight or blood glucose (blood glucose level) between the groups (Figure 5B; Figure 26). However, diabetic GR treated with Wnt inhibitors showed no significant differences in body weight or blood glucose (blood glucose level) between the groups (Figure 5B). ECKO A significant decrease in kidney weight was observed in diabetic and control mice (Figure 5B). Wnt inhibition obviously improved the relative fibrosis area, relative collagen deposition, and tubular damage in diabetic control mice; this effect was consistent with the diabetic GR. ECKO In diabetic control mice, the effects were less pronounced but still significant (Figure 5C). Wnt inhibition was not observed in non-diabetic control mice or non-diabetic GR mice. ECKO Wnt inhibition did not result in significant changes in the renal fibrogenic phenotype or β-catenin levels in diabetic GR mice compared to their untreated non-diabetic controls (FIG. 27). ECKOWnt inhibition suppressed the expression of Snail1 and HIF1α and significantly increased the level of PPARα in the kidneys of diabetic GR mice (Figure 28). Similar effects were observed in isolated ECs (Figure 29). Wnt inhibition also significantly suppressed EndMT (CD31 / αSMA double positive cells) in diabetic control mice; this effect was consistent with the diabetic GR mice. ECKO The effect was less pronounced in mice (Figure 30). However, Wnt inhibition reduced both control and diabetic GR ECKO The levels of EMT (E-cadherin / αSMA double positive cells) were significantly reduced in mice ( FIG. 30 ).

[0107] Example 7. Metabolic reprogramming by loss of endothelial GR accelerates renal fibrosis It is becoming increasingly recognized that defects in central metabolism contribute to renal fibrosis. 32、41 Defects in fatty acid (FA) metabolism in ECs lead to EndMT events 42 To investigate whether FA metabolism was perturbed in our model, we radiolabeled [ 14 [C] palmitate uptake experiments were performed and we observed that FA uptake was higher in isolated ECs from diabetic kidneys of a more fibrotic strain (diabetic CD-1) compared to kidney ECs from a less fibrotic strain (diabetic C57BL / 6). Administration of a Wnt inhibitor suppressed FA uptake in ECs from CD-1 mice (Figure 31A). Diabetic GR ECKO Renal ECs from both diabetic and DKO mice showed higher FA uptake compared to those from diabetic control littermates (Figure 6A). Radiolabeled [ 14 C] from palmitic acid 14 FA oxidation (FAO) was also assessed by measuring [CO2] release. FAO was reduced in isolated kidney ECs from diabetic CD-1 mice, and Wnt inhibition was able to restore the levels of FAO (Figure 31B). ECKOCultured renal ECs from diabetic DKO mice and diabetic control littermates showed reduced FAO levels (Figure 6B). Furthermore, gene expression of fatty acid transporter proteins 1 and 4 (FATP1 and FATP4) was significantly increased in control and GR mice. ECKO was unchanged in both nondiabetic and diabetic ECs isolated from mouse kidneys; however, gene expression levels of another fatty acid transporter, CD36, were lower in diabetic mice of both genotypes compared to nondiabetic controls (Figure 32).

[0108] In the next set of experiments, diabetic CD-1 mice were treated for 4 weeks with the fatty acid (FA) synthase inhibitor C75, the fatty acid oxidation (FAO) inhibitor etomoxir, the PPARα agonist fenofibrate, or the cholesterol-lowering drug simvastatin. Fenofibrate and C75 ameliorated the fibrogenic phenotype, whereas etomoxir exacerbated fibrosis. Simvastatin treatment did not produce a significant suppression of the level of fibrosis (Figure 33A). Fenofibrate and C75 restored the level of GR protein in CD31-positive cells, whereas etomoxir suppressed it and simvastatin had no substantial effect (Figure 33B). Fenofibrate and C75 downregulated fibronectin and αSMA mRNA levels, whereas etomoxir upregulated this mRNA, and simvastatin did not cause significant changes in the gene expression levels of fibronectin or αSMA in diabetic kidneys (Figure 33C). These FA modulators did not result in significant differences in blood glucose levels (Figure 33D). In diabetic CD-1 mice, etomoxir treatment reduced [ 14 C75 and fenofibrate significantly suppressed FAO levels, as measured by CO2 release, and CPT1a levels, and induced β-catenin expression, whereas C75 and fenofibrate increased FAO levels, increased CPT1a expression, and suppressed β-catenin levels (Figures 33E-33F).

[0109] Etomoxir and C75 were administered to nondiabetic and diabetic control littermates and to GRECKO Mice were also tested (Figures 6C-6D; Figure 34; Figure 35). Non-diabetic or diabetic control littermates and GR ECKO There were no significant differences in body weight, blood glucose, or kidney weight in mice after treatment with etomoxir or C75 (Figure 6C; Figure 34). Data from kidney ECs revealed that etomoxir caused a significant suppression of FAO, and C75 restored FAO in non-diabetic control littermates (Figure 34). However, diabetic GR ECKO In renal ECs from mice, etomoxir caused a significant suppression of FAO that C75 could not rescue (Figure 6c). Etomoxir treatment significantly suppressed FAO in diabetic control and diabetic GRs. ECKO C75 accelerated the renal fibrotic phenotype, suppressed CPT1a levels, and increased β-catenin expression levels in the kidneys of diabetic mice. C75 treatment obviously abolished the renal fibrotic phenotype, restored CPT1a, and completely reduced β-catenin levels in the kidneys of diabetic control mice. These effects were consistent with GR. ECKO This was also observed in mice, but to a lesser extent (Figure 6D). Etomoxir reduced the inflammatory response in nondiabetic controls and GR mice. ECKO Etomoxir slightly increased the level of renal fibrosis in diabetic control mice, which could be attenuated by C75 (Figure 35). Etomoxir reduced the expression of CPT1a but increased the expression of β-catenin in diabetic control mice; C75 was able to substantially reverse both of these effects. ECKO A similar pattern was observed in mice, although to a lesser extent (FIG. 6E).

[0110] Example 8. GR loss-associated EndMT disrupts central metabolism and induces mesenchymal transformation in renal tubular epithelial cells This in vivo data indicates that GR ECKOThese results suggest that diabetic mice exhibit enhanced EndMT and EMT in their diabetic kidneys. Overexpression of GR suppressed TGFβ2-stimulated increases in αSMA, collagen I and β-catenin expression in HUVECs (Figure 36). To test whether endothelial GR deficiency affects the mesenchymal program and causes central metabolic defects in nearby epithelial cells, we analyzed the effect of conditioned medium from GR knockdown HUVECs on the mesenchymal phenotype of HK-2 cells (Figure 7A). Conditioned medium (CM) from GR siRNA-transfected HUVECs reduced E-cadherin protein levels and increased αSMA, TGFβR1 and β-catenin protein levels in HK-2 cells compared to medium from scrambled siRNA-transfected HUVECs (Figures 7B-7C). Treatment with CM from GR siRNA-transfected HUVECs caused a significant decrease in the levels of FAO, oxygen consumption rate, and cellular ATP levels in HK-2 cells (Figures 7D-7F). TECs incubated with CM from GR siRNA-treated HUVECs showed significantly downregulated mRNA expression of the FAO-responsive genes Cpt1a, Cpt2, Pparα, and Pgc1α compared with TECs incubated with CM from control siRNA-treated HUVECs (Figure 7G).

[0111] To confirm these in vitro results, diabetic control and diabetic GR ECKO We isolated primary ECs from mice and analyzed the contribution of GR-deficient ECs to mesenchymal activation in renal tubular epithelial cells (TECs). ECKO CM from isolated cultured ECs from kidneys of diabetic and control littermates was transferred to cultured TECs from diabetic control mice (Figure 7H). This treatment with CM from GR-deficient cells caused significant suppression of E-cadherin, CPTla and PPARα, and induced protein levels of αSMA, TGFβR1 and active β-catenin in TECs (Figure 7I). Furthermore, diabetic GR ECKOCM obtained from mouse-derived ECs significantly downregulated the mRNA expression of FAO-responsive genes Cpt1a, Cpt2, and Pparα ( Fig. 7J ).

[0112] The above examples demonstrate the important role of endothelial GR in regulating fibrogenic processes in mouse models of diabetic kidney disease. The results demonstrate that endothelial GR regulates mesenchymal transdifferentiation processes by affecting the control of FA metabolism and canonical Wnt signaling in the kidneys of diabetic mice. From these data, it is clear that GR loss is one of the catalysts of renal fibrosis in diabetes, which causes the disruption of cytokine and chemokine homeostasis by upregulating canonical Wnt signaling. Ultimately, these processes alter the metabolic switch in favor of defective FA metabolism and associated mesenchymal activation in TECs.

[0113] Metabolic reprogramming in ECs is a key event in myofibroblast formation, proliferation and fibrosis progression in the diabetic kidney 12、17、41、43、44 The data suggest that GR deficiency is a critical step in the metabolic reprogramming of renal ECs. Although bilateral adrenalectomy markedly suppressed corticosterone in all mice tested, this global loss of systemic steroid signaling was not sufficient to alter the course of fibrosis in diabetic mice, suggesting that tissue-specific effects of targeted loss of GR in the endothelium supersede its systemic effects; this phenomenon requires further study. ECKOChanges in cytokine levels in the plasma of mice include elevated levels of proinflammatory cytokines (IL-1β, IL-6, and IL-17) and the anti-inflammatory cytokine IL-10. The role of IL-10 in renal fibrosis in diabetic kidney disease has not been thoroughly investigated so far. However, these data demonstrate that IL-6 is a key proinflammatory cytokine that is elevated in conditions of endothelial GR suppression. Neutralization of IL-6 in diabetic mice completely reversed the renal fibrotic phenotype, suggesting an important profibrotic role for IL-6 in diabetes.

[0114] Recently, it has been demonstrated that loss of endothelial GR leads to upregulation of canonical Wnt signaling 27 GR has been shown to exert its anti-inflammatory effects by targeting the NF-kB signaling pathway. 45 However, GR also targets canonical Wnt signaling in ECs independent of its classical target NF-kB. 27、45 Inhibition of Wnt signaling in ECs may prove to be a valuable therapeutic opportunity to combat diabetic kidney disease. The Wnt pathway is known to be a key contributor to renal fibrosis, and activated canonical Wnt signaling contributes to the disruption of cytokine and chemokine homeostasis. 39、46、48 These data demonstrate that higher levels of GR-deficient canonical Wnt signaling are associated with the induction of mesenchymal and fibrogenic markers.

[0115] To further test the therapeutic potential of Wnt inhibition, we used a small molecule - LGK974. Wnt inhibition clearly suppressed canonical Wnt signaling, substantially ameliorated the fibrogenic phenotype in a mouse model of diabetic kidney disease, and restored endothelial GR levels. These data suggest that sustained suppression of canonical Wnt signaling in ECs is one mechanism by which GR exerts its antifibrotic effects. Notably, this effect is probably due to the fact that Wnt inhibition was able to suppress the EMT process in other cell types (TECs) but not alleviate the EndMT process. ECKO This was less clear in mice. Cumulatively, these data suggest that the endothelial GR is a key anti-EndMT molecule.

[0116] There are limited studies investigating lipid metabolism in kidney cells 49、50 However, it is becoming increasingly important that defects in central metabolism contribute to diabetic kidney disease. 32、41 , There are several reports indicating that altered cytokine levels can affect renal lipid metabolism in diabetic kidney disease. 51、52 Clinical observations indicate a potential association between lipid levels and kidney disease. 53 , Lipid control appears to be important in the prevention and treatment of diabetic kidney disease. 49、50 Here, we aimed to elucidate the contribution of lipid metabolism in ECs to the regulation of diabetic kidney disease.

[0117] DKO mice are Apoe - / - showed worsening atherosclerosis compared to mice, which was not explained by differences in plasma lipid levels 25 This observation prompted us to evaluate the diabetic phenotype in these animals. Data showed that hypercholesterolemia is associated with GR ECKOIt is clear that hypercholesterolemia worsens the severity of renal fibrosis in mice, suggesting that hypercholesterolemia affects EC metabolism and contributes to renal fibrosis. However, similar to available clinical data, the cholesterol-lowering drug simvastatin did not improve the severity of renal fibrosis in this mouse model of diabetic kidney disease. Interestingly, fibrates are a class of drugs that treat hypertriglyceridemia with residual elevation of non-HDL cholesterol. However, the role of fibrates in patients with diabetic kidney disease remains to be elucidated. 50、63 .

[0118] We assessed the contribution of endothelial GR loss-linked Wnt activation and its association with defects in FA metabolism. Disruption of endothelial FA metabolism contributes to EndMT activation in diabetic kidneys. 8、31、64 FAO activation caused a marked suppression of fibrosis by restoring endothelial GR levels in diabetic mice. In contrast, FAO inhibition caused accelerated fibrosis by decreasing the levels of endothelial GR in diabetic control mice, suggesting that endothelial GR is a key protein for the action of FAO modulators. These data clearly suggest that the antifibrotic effect of C75, an FAO activator, depends on endothelial GR and, by extension, EC is required for the antifibrotic action of this drug.

[0119] diabetes gr ECKOWhen conditioned medium from GR-depleted ECs from mice was transferred to cultured TECs from diabetic control kidneys, we observed induction of mesenchymal markers, activation of TGFβ and classical Wnt signaling, and concomitant suppression of epithelial cell markers. These findings suggest that EndMT leads to activation of EMT processes in diabetes. GR-depleted cells have higher levels of TGFβ-smad3 and classical Wnt signaling, which are associated with disrupted levels of plasma cytokines, and suppression of FAO. The cumulative effect of these metabolic changes leads to activation of mesenchymal transformation in ECs, which may exert paracrine effects on nearby TECs. The functional importance of GR protein in EC homeostasis is illustrated in Figure 8.

[0120] GR agonists such as dexamethasone activate GR signaling in all cell types; however, in diabetes, dexamethasone intervention is undesirable because it leads to severe and predictable worsening of hyperglycemia. These data highlight a regulatory role of GR in ECs.

[0121] In conclusion, these findings highlight a regulatory role of GR on EndMT in the diabetic kidney, mediated by control over canonical Wnt signaling and associated defective FA metabolism.The present study provides insight into the biology of GR and its key role in renal fibrosis and diabetes.

[0122] The following are the methods used in the above examples.

[0123] Reagents and antibodies. Rabbit polyclonal anti-GR (SAB4501309), mouse monoclonal anti-αSMA (catalog number: A5228) and mouse monoclonal anti-β-actin (AC-74) (A2228) antibodies were obtained from Sigma (St Louis, MO). Anti-TGFβR1 (ab31013), PPARα (ab215270), mouse monoclonal anti-vimentin (RV202) (ab8978), rabbit polyclonal anti-αSMA (ACTA2) (ab5694), anti-HIFlα (ab516008) and goat polyclonal anti-Snail1 (ab53519) antibodies were purchased from Abcam (Cambridge, UK). Mouse anti-β-catenin antibody (610154) was purchased from BD Biosciences. Carnitine palmitoyltransferase 1a (CPT1a) (12252), rabbit polyclonal anti-E-cadherin antibody (24E10) (3195) and rabbit non-phospho (active) β-catenin (8814) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-HSP90 was purchased from BD Biosciences (610419). In vivo mouse IL-6 IgG neutralizing antibody and control IgG antibody were purchased from Bio X Cell. Fluorescent-, Alexa fluor 647-, and rhodamine-conjugated secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). TGFβ2, IL-1β and recombinant TNFα and TGFβ neutralizing antibodies were purchased from PeproTech (Rocky Hill, NJ). Etomoxir, C75 and Wnt inhibitor (LGK974) were purchased from Cayman Chemical (Ann Arbor MI).

[0124] Animal experiments. All experiments were performed according to protocols approved by the Yale University School of Medicine Animal Care and Use Committee and were in compliance with the National Institutes of Health (NIH) guidelines for laboratory animal care. Mice were housed in an environment with room temperature of 68–79°F and humidity of 30–70%. They were exposed to a 12-h light / dark cycle. Mice lacking the endothelial glucocorticoid receptor (GR) (GRECKO (known as IL-1, IL-2, and mice lacking this receptor (DKO) on an Apoe null background were generated as previously described. 25 these mice were on a C57BL / 6 background. Diabetes was induced in 10-week-old male mice by intraperitoneal (IP) administration of 50 mg / kg streptozotocin (STZ) in 10 mmol / L citrate buffer (pH 4.5) for 5 consecutive days. A Wnt inhibitor (LGK974) was administered to 16-week-old STZ-treated diabetic mice at a dose of 5 mg / kg, 6 times a week for 8 weeks. 40 Etomoxir (20 mg / kg) and C75 (15 mg / kg) were ECKO and control littermate mice were administered intraperitoneally (IP) three times a week for three weeks.

[0125] Diabetes was induced in CD-1 mice using a single IP dose of 200 mg / kg STZ.

[0126] In one experiment, male mice were randomly assigned to one of four groups 16 weeks after induction of diabetes: (i) untreated, (ii) fenofibrate (100 mg / kg), (iii) simvastatin (40 mg / kg), or (iv) Wnt inhibitor (LGK974; 5 mg / kg). In each case, mice were treated for 4 weeks and compared to untreated diabetic CD-1 mice. In another experiment, male diabetic CD-1 mice were randomly assigned to one of three groups: (i) untreated (vehicle), (ii) etomoxir (20 mg / kg), or (iii) C75 (15 mg / kg); in each case, mice were treated (IP) 3 times / week for a total of 4 weeks.

[0127] In both non-diabetic and diabetic mice, IL-6 IgG and control IgG were IP injected at a dose of 3 mg / kg, 3 times / week for a total of 4 weeks. All mice had free access to food and water during the experiment. Blood was collected by retro-orbital plexus bleeding during the experiment. Blood glucose (blood glucose level) was measured by glucose strips. Urinary albumin levels were measured using a mouse albumin ELISA kit (Exocell, Philadelphia, PA).

[0128] Tissues and blood were collected at the time of sacrifice. Some kidneys were minced and stored at -80°C for gene expression and protein analysis. Other kidneys were immediately placed in optimal cutting temperature (OCT) compound for frozen sections or 4% paraformaldehyde for histological staining.

[0129] Bilateral adrenalectomy. Diabetic mice injected with STZ for 16 weeks (24 weeks old) and nondiabetic CD-1 and C57BL / 6 mice were used for adrenalectomy and sham surgery. To reduce stress responses, animals were handled daily during the final week before the experiment. Buprenorphine was used as an analgesic. The first dose was administered subcutaneously at a dose of 0.05 mg / kg 30 min before surgery, then every 12 h for 72 h. For the first 24 h after surgery, mice were given drinking water containing 0.9% saline to counter the effects of mineralocorticoid removal. Adrenalectomized animals were sacrificed 4 weeks after surgery. Blood was collected between 9:00 and 10:00 a.m. Corticosterone levels were measured using an ELISA from Cayman Chemical (Ann Arbor MI).

[0130] Unilateral ureteral obstruction (UUO) mouse model. Surgical procedures for UUO were performed as previously described. 19Briefly, mice were anesthetized with isoflurane (3%-5% for induction and 1%-3% for maintenance). The left ventral side of the mouse was shaved, a vertical incision was made in the skin with a scalpel, and the skin was peeled off. A second incision was made through the peritoneum to expose the kidney. The left ureter was ligated twice with surgical silk thread 15 mm below the renal pelvis, and then the ureter was cut between the two ligatures. The ligated kidney was then gently returned to its correct anatomical position, and sterile saline was added to compensate for fluid loss. The incision was sutured, and the mice were caged individually. Buprenorphine was used as an analgesic. The first dose was administered subcutaneously 30 min before surgery, then every 12 h for 72 h at a dose of 0.05 mg / kg. Ten days after UUO, the mice were sacrificed, and kidney and blood samples were taken after perfusion with PBS. For all experiments with this model, the contralateral kidney was used as a non-fibrotic control.

[0131] Blood pressure measurements. Measurements were performed using the tail-cuff method according to the manufacturer's instructions. Briefly, mice were trained for 5 days before blood pressure was measured. After placing the mouse in a restraining platform maintained at 33-34 °C, the tail was passed through an optical sensor and compressed with the cuff. The instrument automatically measured blood pressure, which was repeated 10 times. Data are presented as the average of 10 measurement cycles.

[0132] Lipid analysis. Mice were fasted for 12–15 h, and blood was collected by retro-orbital plexus puncture. Whole blood was spun down and plasma was stored at −80°C. Total cholesterol and triglyceride levels were measured enzymatically by Wako and Sigma kits, respectively, according to the manufacturer's instructions.

[0133] Morphological evaluation. The relative area of ​​mesangial matrix was evaluated using a point counting method. We analyzed PAS-stained glomeruli from each mouse using a digital microscope screen grid containing 540 (27 × 20) points. Masson's trichrome stained images were evaluated by ImageJ software to estimate the fibrotic area.

[0134] Sirius red staining. Deparaffinized sections were incubated with picrosirius red solution for 1 h at room temperature. Slides were washed twice with acetic acid solution for 30 s per wash. Slides were then dehydrated three times in absolute alcohol, cleared in xylene, and embedded in synthetic resin. Sirius red staining was analyzed using ImageJ software, and the fibrotic area was quantified.

[0135] Immunohistochemistry. Paraffin-embedded kidney sections (5 μm thick) were deparaffinized, rehydrated (4 times 2 min in xylene; 2 times 1 min in 100% ethanol; 1 min in 95% ethanol; 45 s in 70% ethanol; 1 min in distilled water) and antigen retrieved in 10 mM citrate buffer (pH 6) at 98 °C for 60 min. To block endogenous peroxidase, all sections were incubated in 0.3% hydrogen peroxide for 10 min. Immunohistochemistry was performed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Mouse anti-β-catenin antibody (1:100) and CPT1a (Abnova; H00001374-D01P; 1:100) antibodies were used. In negative controls, the primary antibody was omitted and replaced with blocking solution.

[0136] Immunofluorescence. Frozen kidney sections (5 μm) were used for immunofluorescence; double positive labeling with CD31 / αSMA (1:100 / 1:500), CD31 / TGFβR1 (1:100 / 1:500) and E-cadherin / αSMA (1:500 / 1:500) was measured. Briefly, frozen sections were dried and placed in acetone for 10 min at -30°C. After drying, sections were washed twice for 5 min in phosphate buffered saline (PBS) and then blocked for 30 min at room temperature in 2% bovine serum albumin / PBS. Sections were then incubated with primary antibody (1:100) for 1 h and washed three times in PBS (5 min each). Sections were then incubated with secondary antibodies for 30 min, washed three times (5 min each) with PBS, and mounted with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Immunolabeled sections were analyzed by fluorescence microscopy. For each mouse, photographs at original magnification of x400 were obtained from six different areas and quantification was performed.

[0137] Detection of EndMT and EMT. Frozen sections (5 μm) were used for detection of EndMT and EMT. Cells undergoing EndMT were detected by double positive labeling for CD31 (1:100) and αSMA (1:500) and / or TGFβR1 (1:500). Cells undergoing EMT were detected by double positive labeling for E-cadherin (1:500) and αSMA (1:500). Sections were analyzed and quantified by fluorescence microscopy.

[0138] Isolation of endothelial cells. Endothelial cells were isolated from kidneys of non-diabetic and diabetic mice using a standardized kit (Miltenyl Biotech, USA) according to the manufacturer's instructions. Briefly, kidneys were isolated and minced into small pieces. Single-cell suspensions were made using a series of enzymatic reactions by treating the tissue with trypsin and type I collagenase solutions. The pellet was lysed with CD31 magnetic beads, and CD31-labeled cells were separated with a magnetic separator. Cells were further purified with columns, cell counts were counted with a hemocytometer, and cells were seeded on Petri dishes coated with 0.1% gelatin. Cell purity was measured by flow cytometry (BD FACSDiva) using both PE-conjugated CD31 (BDB553373) (1:100) and FITC-conjugated CD45 (BDB553079) (1:100).

[0139] Isolation of renal TECs. After sacrifice, diabetic GR ECKO Kidneys were removed from rats and control littermates and perfused with collagenase (10 mL) followed by digestion with collagenase type II (2 mg / mL). After digestion, the cortical region of the kidney was used for further processing. The cortical region of the kidney was minced and further digested in collagenase buffer for an additional 5 min at 37 °C with rotation to release cells. The digested tissue and cell suspension was passed through a 70 μm cell strainer, centrifuged at 50 g for 5 min, and washed twice in PBS to collect TECs. The isolated TECs were seeded on collagen-coated Petri dishes and cultured in renal epithelial cell medium (C-26130, PromoCell) supplemented with growth factors for TEC proliferation.

[0140] Cellular bioenergetics analysis. FAO-associated oxygen consumption rate (OCR) was studied using extracellular flux analysis (Seahorse XFe96, Agilent Technologies). On the day of the assay, the substrate-limited medium was replaced with Krebs-Henseleit buffer assay medium supplemented with 0.2% carnitine for 1 h at 37 °C without CO2. Finally, BSA or 200 mM palmitic acid-BSA FAO substrate was added just before starting the assay. After the assay, for data normalization, proteins were extracted from the wells with 0.1% NP-40-PBS solution and quantified with a bicinchoninic acid protein assay (Thermo Fisher Scientific). OCR was determined by quantifying the Hoechst staining of each well, normalizing the measurements to cell number. 65 .

[0141] ATP Measurement. ATP content was determined using an ATP colorimetric assay kit (Biovision) according to the manufacturer's instructions.

[0142] RNA isolation and qPCR. Total RNA was isolated using standard Trizol protocols. RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad) and qPCR was performed on a Bio-Rad C1000 Touch thermal cycler using the resulting cDNA as well as the qPCR master mix and gene-specific primers. A list of mouse primers used is shown in Table 1. Results were quantified using the delta-delta cycle threshold (Ct) method (ΔΔCt). All experiments were performed in triplicate and 18S was used as an internal control. [Table 1]

[0143] Western blot. Protein lysates were boiled in sodium dodecyl sulfate (SDS) sample buffer at 94°C for 5 min. After centrifugation at 17,000xg for 10 min at 4°C, the supernatants were separated on 6%-12% SDS polyacrylamide gels and blotted onto PVDF membranes (Immobilon, Bedford, MA) by the semi-dry method. After blocking with TBS (Tris-buffered saline containing 0.05% Tween 20) containing 5% bovine serum albumin (BSA), the membranes were incubated overnight at 4°C with each primary antibody (GR: 1:1000; anti-TGFβR1: 1:500; anti-αSMA: 1:500; anti-vimentin: 1:2000, anti-β-catenin: 1:500, anti-β-actin 1:10,000) in TBS containing 5% BSA. Protein bands were visualized using an Odyssey Infrared Imaging System (LI-COR Biotechnology) and an enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology, Rockland IL) using an ImageQuant LAS400 (GE Healthcare Life Sciences, Uppsala, Sweden). Densitometry was performed using ImageJ software (NIH).

[0144] In vitro experiments and siRNA transfection. HUVECs were used at passages 4–8 and cultured in Endothelial Basal Medium-2 medium containing growth factors and 10% serum. Human GR-specific siRNA (Invitrogen) was used at a concentration of 100 nM for 48 h to effectively knock down GR. Cells were treated with or without TGFβ2 (10 ng / ml) for 48 h and harvested for Western blot analysis. Some transfected cells were treated with fenofibrate (1 μM) and etomoxir (40 μM) for 48 h. In the second set of experiments, human HK-2 cells were cultured in DMEM and Keratinocyte-SFM (1X) medium (Life Technologies Green Island NY). When cells reached 70% confluence, conditioned medium from control siRNA- and GR siRNA-transfected HUVECs was added to HK-2 cell cultures.

[0145] Fatty acid uptake. Cultured isolated kidney endothelial cells were treated with 2 μCi of 14 C]-palmitic acid. 14 C]-palmitic acid incorporation was measured by liquid scintillation counting.

[0146] Fatty acid oxidation. Cultured isolated kidney endothelial cells were incubated with 0.75 mmol / L palmitic acid (2% free fatty acid-conjugated to free BSA / 2 μCi / mL [ 14 The cells were incubated for 2 hours with a medium containing [C] palmitic acid. 1 mL of the culture medium was transferred to a sealable tube, the cap of which was fitted with a Whatman disk filter. The medium was then acidified with 60% perchloric acid to remove the trapped leukocytes. 14 CO2 was allowed to evolve, and the radioactivity adsorbed to the disk filters was then quantified by liquid scintillation counting.

[0147] Statistical analysis. All values ​​are expressed as the mean ± SEM and analyzed using the GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA) statistical package. One-way ANOVA followed by Tukey's test and two-way ANOVA (Figures 1 and 2) were used to analyze significance when comparing multiple independent groups. Post-hoc tests were performed only if F achieved P<0.05 and there was no significant variation in homogeneity. In each experiment, N represents the number of separate experiments (in vitro) and number of mice (in vivo). Technical replicates were used to ensure the reliability of single values. Data analysis was blinded. Data were considered statistically significant at p<0.05. References

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[0149] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0150] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein.

Claims

1. 1. A method for treating a condition or disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of IL-6 activity, the condition or disease being selected from organ fibrosis, nephropathy, dyslipidemia, hypertension, hyperlipidemia, hypercholesterolemia, cardiovascular disease, peripheral artery disease, atherosclerosis, coronary artery disease, coronary heart disease, and stroke.

2. The method of claim 1, wherein the organ fibrosis is renal fibrosis.

3. The method of claim 1, wherein the nephropathy is diabetic nephropathy.

4. The method of any one of claims 1 to 3, wherein the inhibitor of IL-6 activity is an antibody or an antigen-binding fragment of an antibody.

5. The method of claim 4, wherein the inhibitor is a neutralizing antibody or antigen-binding fragment thereof for IL-6.

6. 5. The method of claim 4, wherein the inhibitor is sirukumab, siltuximab, or olokizumab, or an antigen-binding fragment thereof.

7. The method of claim 4, wherein the inhibitor is an antibody or antigen-binding fragment that targets the IL-6 receptor.

8. 8. The method of claim 7, wherein the inhibitor is tocilizumab or sarilumab, or an antigen-binding fragment thereof.

9. The method of any one of claims 1 to 3, wherein the inhibitor of IL-6 activity results in a blockade of trans-signaling.

10. 10. The method of claim 9, wherein the inhibitor is olamxept (FE999301), or a fragment or variant thereof.

11. The method according to any one of claims 1 to 3, wherein the inhibitor of IL-6 activity is an inhibitor of intracellular signaling.

12. The method of claim 11, wherein the inhibitor is tofacitinib or CpG-stat3 siRNA.

13. The method of any one of claims 1 to 12, further comprising administering to the subject one or more additional agents effective to treat the condition or disease.

14. 14. The method of claim 13, wherein the one or more additional agents comprise one or more cholesterol-lowering agents, antihypertensive agents, anti-inflammatory agents, antithrombotic agents, anticoagulants, renin-angiotensin-aldosterone system inhibitors (RAAS inhibitors), beta-adrenergic blockers, calcium channel blockers, hypoglycemic agents, and / or Wnt inhibitors.

15. 15. The method of claim 14, wherein the cholesterol-lowering drug comprises a statin, a fibrate, and / or an inhibitor of proprotein convertase subtilisin / kexin type 9.

16. 15. The method of claim 14, wherein the antihypertensive therapy comprises an angiotensin-converting enzyme (ACE) inhibitor and / or an angiotensin II receptor blocker (ARB).

17. 15. The method of claim 14, wherein the hypoglycemic agent comprises metformin, insulin, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide 1 (GLP-1), and / or a sodium / glucose cotransporter 2 (SGLT2) inhibitor.

18. The Wnt inhibitor has the formula (I): 【Chemistry 1】 [In the formula, X １ and X ２ is selected from N and CR; X ３ , X ４ , X ５ and X ６ is N and the other is selected from N and CR; X ７ , X ８ , X ９ and X １０ is N and the other is selected from N and CR; X １１ , X １２ , X １３ and X １４ is N and the other is selected from N and CR; R is independently selected at each occurrence from hydrogen, halo, cyano, methyl, difluoromethyl, and trifluoromethyl.

15. The method of claim 14, comprising a compound having the structure: or a pharma- ceutically acceptable salt thereof.

19. The method of any one of claims 1 to 18, wherein the subject is a human.

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