IL-6 inhibitors for use as therapeutics for nephropathy

Abstract

Methods and compositions are provided for treating diseases or conditions (e.g., organ fibrosis, kidney disease) by administering an inhibitor 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 on February 23, 2022, the disclosure of which is incorporated herein by reference in its entirety. Government funding

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

[0003] The present disclosure generally relates to the use of IL-6 inhibitors for treating diseases and disorders such as organ fibrosis or kidney disease. Sequence Listing

[0004] This application contains a sequence list that has been submitted electronically in XML file format, and the entire content is incorporated herein by reference. The XML copy was created on February 20, 2023, named 251609_000088_SL.xml, with a size of 23,686 bytes. Background Art

[0005] Approximately one-third of patients with diabetes will develop diabetic nephropathy (DN), the leading cause of end-stage renal disease (ESRD), which causes more than 950,000 deaths worldwide each year1,2. In the past two decades, no new drugs have been approved specifically to prevent diabetic nephropathy or improve renal function3. This lack of progress has been attributed in part to an inadequate understanding of the mechanisms of progressive renal insufficiency. Furthermore, this knowledge gap makes the available treatment options for these patients suboptimal. A deeper understanding of the pathogenesis of diabetic nephropathy is urgently needed to facilitate the development of new effective and safe therapies that target the early stages of diabetes, before kidney damage becomes irreversible.

[0006] Diabetic nephropathy is characterized by excessive deposition of extracellular matrix, loss of the capillary network, and accumulation of fibrillar collagen, activated myofibroblasts, and inflammatory cells. 4,5 In renal fibrosis, myofibroblasts are considered to be an activated fibroblast phenotype that promotes fibrosis. 5,6 There are six widely 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 tubular epithelial cells via epithelial-to-mesenchymal transition (EMT), and (6) from mesenchymal cells transformed from endothelial cells (EC) via endothelial-to-mesenchymal transition (EndMT). 7,8 .

[0007] Among the different sources of matrix-producing fibroblasts, mesenchymal cells derived from ECs via EndMT 7,8 An important source of myofibroblasts in multiple organs including the kidney 9 EndMT is characterized by the loss of endothelial cell markers, including cluster of differentiation 31 (CD31), and the gain of expression of mesenchymal proteins, including α-smooth muscle actin (αSMA), vimentin, and fibronectin. 7,8 .

[0008] ECs are key contributors to neovascularization in health- and life-threatening diseases 10 Disturbance in EC central metabolism contributes to the disease phenotype 11,12 Carnitine palmitoyltransferase 1a (CPT1a)-mediated fatty acid oxidation (FAO) regulates EC proliferation in the stem of outgrowth vessels 13-15 ECs use metabolites / precursors for epigenetic regulation of their subtype differentiation and maintain cross-talk via metabolites released by other cell types 10,15 Notably, EndMT leads to changes in endothelial cell metabolism, which is an area of ​​active research. 16,17 For example, EndMT-derived mesenchymal cells reprogram their metabolism and exhibit defective fatty acid metabolism. 17 .

[0009] The contribution of EndMT to renal fibrosis has been analyzed in several mouse models of chronic kidney disease. 5-7,18,19 Zeisberg et al. conducted pioneering experiments and reported that approximately 30–50% of fibroblasts in the kidneys of mice with unilateral ureteral obstruction (UUO) co-expressed the EC marker CD31 as well as markers of fibroblasts and myofibroblasts, such as fibroblast-specific protein-1 (FSP-1) and αSMA. 19 Complete conversion of ECs to a mesenchymal cell type is not required, as an intermediate cell type is sufficient to activate profibrogenic pathways. EndMT may induce profibrogenic signaling in neighboring cells through autocrine and / or paracrine mechanisms, thereby contributing to overall renal fibrosis 6,20,21 Therefore, targeting EndMT may have therapeutic potential for the treatment of renal fibrosis. 6,19,22 . Summary of the invention

[0010] The glucocorticoid receptor (GR) is a nuclear hormone receptor ubiquitously expressed in most cell types and is important in many states of health and disease. The glucocorticoid receptor (GR) mediates the actions of steroid hormones in a variety of tissues, including the kidney. The role of glucocorticoids in cardiovascular and renal diseases is complex. The inventors of the present disclosure identified a role for endothelial GR in sepsis. 23 and atherosclerosis 25 Endothelial GR acts as a negative regulator of vascular inflammation in models. Loss of endothelial GR leads 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 evolution of renal fibrosis.

[0011] Based on the work presented herein, the inventors have shown that mice lacking endothelial GR exhibit accelerated renal fibrosis under diabetic and non-diabetic conditions. This exacerbated fibrosis is associated with abnormal chemokine and cytokine reprogramming, increased Wnt signaling, and inhibited fatty acid oxidation. The present disclosure indicates that endothelial GR is a key molecule involved in regulating the fibrotic process in the kidney.

[0012] One aspect of the present disclosure provides a method for treating a condition or disease of a subject in need thereof. The method may include administering to the subject an inhibitor of a therapeutically effective amount of IL-6 activity, wherein the condition or disease is selected from organ fibrosis, nephropathy, dyslipidemia, hypertension, hyperlipidemia, hypercholesterolemia, cardiovascular disease, peripheral arterial 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.

[0013] In some embodiments of the methods of treatment 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 antigen-binding fragment targeting an IL-6 receptor, such as, but not limited to, tocilizumab or sarilumab, or an antigen-binding fragment thereof.

[0014] In some embodiments of the treatment methods described herein, the inhibitor of IL-6 activity provides a blockade of trans-signaling, such as, but not limited to, olamkicept (FE999301) or a fragment or variant thereof.

[0015] 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.

[0016] In various embodiments of the treatment methods described herein, the methods further comprise administering to the subject one or more additional agents effective for treating the condition or disease.

[0017] In some embodiments, the one or more additional agents include one or more of the following: cholesterol-lowering drugs, blood pressure-lowering therapies, anti-inflammatory agents, anti-thrombotic agents, anticoagulants, inhibitors of the renin-angiotensin aldosterone system (RAAS inhibitors), beta-adrenergic blockers, calcium channel blockers, hypoglycemic drugs, and / or Wnt inhibitors.

[0018] In some embodiments, the cholesterol-lowering drug comprises a statin, a fibrate, and / or an inhibitor of proprotein convertase subtilisin / kexin type 9 (PCSK9).

[0019] In some embodiments, the blood pressure lowering therapy comprises angiotensin converting enzyme (ACE) inhibitors and / or angiotensin II receptor blockers (ARBs).

[0020] In some embodiments, the blood glucose lowering drugs include metformin, insulin, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide 1 (GLP-1) and / or sodium / glucose co-transporter 2 (SGLT2) inhibitors.

[0021] In some embodiments, the Wnt inhibitor includes a compound having a structure according to Formula (I): wherein 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 them is N, and the others are selected from N and CR; X 11 , X 12 , X 13 and X 14 One of them is N, the others are selected from N and CR, and R, at each occurrence, is independently selected from hydrogen, halogen, cyano, methyl, difluoromethyl and trifluoromethyl, or a pharmaceutically acceptable salt thereof.

[0022] In various embodiments of the therapeutic methods described herein, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A-1B shows that loss of the endothelial glucocorticoid receptor (GR) leads to a fibrogenic phenotype in the kidneys of diabetic mice. Figure 1 A) Immunofluorescence analysis was performed in the 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 is 400x. Scale bar, 50 μm. N = 7 biologically independent mice per group, combined from 3 separate experiments. Quantification of GR / CD-31 double-labeled cells is shown. Data are mean ± SEM. ( Figure 1 B) 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 6 biologically independent mice per group. Three independent experiments were performed. Densitometry analysis of 6 samples per group is shown and normalized to β-actin. mRNA expression of 6 independent cell culture samples was analyzed and normalized to 18S. Two independent experiments were performed in triplicate. Data are mean ± SEM. For all groups, statistical significance analysis was performed using two-way ANOVA. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0024] Figure 2 A-2J shows that loss of endothelial GR exacerbates renal fibrosis in diabetic mice. Figure 2 A) Schematic diagram showing the expression of GR fl / fl; Tie1 Cre- (control) GRfl / fl; Tie1 Cre+ (GR ECKO ), Apoe - / - GR fl / fl; Tie1Cre-(Apoe - / - ) and Apoe - / - ; GR fl / fl; Tie1 Cre+(DKO) mice were injected with 5 doses of STZ (50 mg / kg IP per day) to induce diabetes and renal fibrosis. Figure 2 Physiological parameters including body weight, blood glucose, kidney weight / body weight, heart weight / body weight, liver weight / body weight, albumin to creatinine ratio (ACR), plasma triglycerides and plasma cholesterol were measured. N = 6 biologically independent mice per group from 2 separate experiments. ( Figure 2 J) Non-diabetic and diabetic controls, GR ECKO、Apoe - / - Kidneys of DKO and DKO mice were stained with Masson trichrome and Sirius red. Representative images are shown; original magnification is 300 times. Relative area of ​​fibrosis (%) and relative collagen (%) were measured using the ImageJ program. N = 7 biologically independent mice per group from 2 separate experiments. Scale bar is 50 μm. Data are mean ± SEM. For all groups, statistical significance was analyzed using two-way ANOVA. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0025] Figure 3 A-3C shows that IL-6 neutralization rescues renal fibrosis in diabetes. ( Figure 3 A) Plasma IL-6 levels in non-diabetic and diabetic mice were measured by cytokine array (Luminex). N = 5 biologically independent mice per group. ( Figure 3 B) IL-6 was measured in the culture 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 triplicate. Data are mean ± SEM. ( Figure 3 C) Diabetes GR ECKO Neutralization of IL-6 in diabetic mice and diabetic DKO mice. Representative images are shown; original magnification is 300 times. Relative area of ​​fibrosis (%) and relative collagen (%) were measured using the ImageJ program. N=7 biologically independent mice per group. Scale bar is 50 μm. Data are mean ± SEM. For all groups, statistical significance was analyzed using one-way ANOVA and Tukey post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0026] Figure 4 A-4C demonstrates upregulation of Wnt signaling and fibrogenic markers accompanying loss of endothelial GR. Figure 4 A) The results of the study were as follows: ECKO Western blot analysis of vimentin, HIF1α, Snail1, active β-catenin, CPT1a, and PPARα in endothelial cells isolated from the kidney of mice. Endothelial cells were isolated and cultured from 6 biologically independent mice per group. Densitometry calculations of 6 samples per group from 3 independent experimental combinations are shown and normalized to β-actin or HSP90 as indicated. Representative blots are shown. Data are mean ± SEM. ( Figure 4 B) In the diabetic control group, GR ECKO 、Apoe - / -Western blot analysis of αSMA, fibronectin, active β-catenin, and β-catenin in isolated endothelial cells from kidneys of A and DKO mice. Endothelial cells were isolated and cultured from 6 biologically independent mice per group. Densitometry calculations of 6 samples per group from 3 independent experiments are shown and normalized to β-actin or HSP90 as indicated. Data are mean ± SEM. Representative blots are shown. ( Figure 4 C) In non-diabetic and diabetic controls, GR ECKO 、Apoe - / - Immunofluorescence analysis of αSMA / CD31 and TGFβR1 / CD31 was performed in the kidneys of DKO and DKO mice. FITC-labeled αSMA, FITC-labeled TGFβR1, rhodamine-labeled CD31, and DAPI (nuclei) were used. Representative merged images are shown; the original magnification is 300 times. The scale bar is 50 μm. N=7 biologically independent mice per group. Data are mean ± SEM. For all groups, statistical significance was analyzed using one-way ANOVA and Tukey post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0027] Figure 5 A-5C shows that Wnt inhibitor partially abolished diabetic GR ECKO and renal fibrosis in DKO mice. Figure 5 A) shows diabetic controls and GR ECKO Schematic representation of the treatment regimen for Wnt inhibition in mice. Figure 5 B) Physiological parameters including body weight, blood glucose and kidney weight / body weight were analyzed. N = 6 biologically independent mice per group, combined from 2 experiments. ( Figure 5 C) Diabetic controls and GR treated with WntI ECKO Kidneys of mice were stained with Masson's trichrome, Sirius red, and PAS, and immunohistochemical analysis of β-catenin levels was performed. Representative images are shown; original magnification is 300 times. Relative area of ​​fibrosis (%) and relative collagen deposition (%) were measured using the ImageJ program. N = 8 biologically independent mice per group were analyzed, combined from 3 experiments. Scale bar, 50 μm. Data are mean ± SEM. For all groups, statistical significance was analyzed using one-way ANOVA and Tukey post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0028] Figure 6 A-6E shows that metabolic reprogramming caused by loss of endothelial GR exacerbates diabetic nephropathy. Figure 6A) Non-diabetic and diabetic controls, GR ECKO 、Apoe - / - Endothelial cells isolated from kidneys of DKO and DKO mice were radiolabeled 14 C-palmitate uptake analysis. CPM of each sample was calculated. Data were normalized to μg protein. Fold activity is shown. Endothelial cells were isolated and cultured from 6 biologically independent mice per group in triplicate and analyzed. 14 C-palmitic acid uptake. Data are mean ± SEM. ( Figure 6 B) GR ECKO 、Apoe - / - Radiolabeling in endothelial cells isolated from kidneys of DKO and DKO mice 14 C] Palmitic acid oxidation and [ 14 CO2] release. CPM of each sample was counted. Data were normalized to μg protein. Fold activity is presented. Endothelial cells were isolated and cultured from 6 biologically independent mice per group in triplicate and analyzed 14 C-palmitic acid uptake. Data are mean ± SEM. ( Figure 6 C) Body weight, blood glucose, and kidney weight / body weight were measured at the end of these experiments. N = 6 biologically independent mice from 2 experiments. Ex vivo radiolabeled [ 14 C] Palmitic acid oxidation and [ 14 CO2] release. CPM counts were performed for each sample. Isolated endothelial cells from 5 biologically independent mice in each group were analyzed in triplicate. Data are mean ± SEM. ( Figure 6 D) Masson trichrome, Sirius red, and PAS staining were analyzed in kidneys of control, etomoxir-treated, and C75-treated diabetic mice. Representative images are shown; original magnification is 300x. Relative area of ​​fibrosis (%) and relative collagen deposition (%) were measured using the ImageJ program. N = 8 biologically independent mice per group were analyzed from 3 independent experiments. Scale bar, 50 μm. Data are mean ± SEM. ( Figure 6 E) Immunohistochemical analysis for CPT1a and β-catenin. Representative images are shown; original magnification is 300x. Scale bar, 50 μm. N = 6 biologically independent mice per group from 3 independent experiments. Statistical significance was analyzed using one-way ANOVA with Tukey post hoc test for all groups. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0029] Figure 7 A-7J shows that loss of GR in endothelial cells leads to central metabolic reprogramming of tubular cells. Figure 7 A) Conditioned medium experimental design. HUVEC were transfected with scrambled siRNA or GR siRNA; after 6 hours, the medium was replaced and the cells were incubated for 96 hours. The harvested medium was then transferred to HK-2 cells. Figure 7 B) 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 per group were combined from 4 independent experiments. Densitometry analysis relative to β-actin is shown. ( Figure 7 C) Immunofluorescence analysis of E-cadherin and α-SMA expression in TECs treated with conditioned medium. Images from 6 different fields of view were evaluated at magnification. n = 7 independent samples per group. Scale bar 30 μm. ( Figure 7 D) Through [ 14 CO2 release measurement 14 C] Palmitic acid oxidation. CPM was counted and normalized to protein in the well. n = 8 independent samples per group. ( Figure 7 E) Oxygen consumption rate (OCR) in TECs treated with conditioned medium; each data point represents the mean of 8 independent samples. Three independent experiments were performed. OCR was measured in a Seahorse XF96 analyzer. ( Figure 7 F) Cellular ATP measurement. Eight independent samples were analyzed. Figure 7 G) Relative mRNA levels of FAO regulators in conditioned medium treated TECs determined by qRT-PCR. n = 7 independent samples per group performed in triplicate were analyzed. ( Figure 7 H) Experimental design of conditioned medium experiment. ECKO ECs from kidneys of diabetic and control mice were cultured for 96 hours. The culture medium harvested subsequently was transferred to TECs from diabetic control mice. n = 7 per group. ( Figure 7 I) Representative Western blot analysis of αSMA, TGFβR1, active β-catenin, E-cadherin, CPT1a, and PPARα expression. Representative blots of 7 biologically independent samples per group combined from 3 independent experiments are shown. Densitometry calculations were normalized to β-actin or HSP90 as indicated. ( Figure 7 J) Relative mRNA levels of FAO regulatory factors in isolated TEC treated with conditioned medium were determined by qRT-PCR. n = 6 independent replicates per group. Data are mean ± SEM. Figure 7 B-7F, Figure 7 I, using Student's t test (unpaired two-tailed); Figure 7G and Figure 7 J, Statistical significance was analyzed using one-way ANOVA and Tukey post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0030] Figure 8 A graphical representation of the role of GR in endothelial cell homeostasis is shown. GR is essential for endothelial cell homeostasis and its loss leads to disruption of homeostasis through the release of the proinflammatory cytokine IL-6 and aberrant activation of Wnt signaling, resulting in defective fatty acid oxidation and associated mesenchymal activation.

[0031] Fig. 9 A-9C shows the effect of adrenalectomy on renal fibrosis. Adrenalectomized and nonadrenalectomized control and diabetic mice of the CD-1 and C57BL / 6 strains were evaluated 4 weeks after bilateral adrenal surgery. Fig. 9 A) Plasma corticosterone levels were measured in control and diabetic CD-1 and C57BL / 6 mice. n = 7 biologically independent mice per group, combined from 2 separate experiments. ( Fig. 9 B) Blood glucose measurements in adrenalectomized and nonadrenalectomized mice. n = 7 biologically independent mice per group, combined from 2 separate experiments. ( Fig. 9 C) Kidneys from control and diabetic mice with and without adrenalectomy were stained with Masson's trichrome and Sirius red. Representative images are shown; original magnification is 300x. Fibrosis area (%) and relative collagen deposition (%) were measured using the ImageJ program. n = 7 biologically independent mice per group, combined from 2 separate experiments. Scale bar, 50 μm. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. C-CD1: non-diabetic control CD1, DM-CD1: diabetic CD-1, C-BL6: non-diabetic control C57BL / 6, DM-BL6: diabetic C57BL / 6 mice, and ADX: adrenalectomized.

[0032] Fig.10 Determination of renal endothelial cell purity is shown. Upper panel: Flow cytometric cell purity analysis of endothelial cells isolated from kidney. CD31+CD45- cells are shown. Lower panel: Gating strategy in unstained, blocked endothelial cells is shown.

[0033] Fig.11 A-11B shows the analysis of GR protein and mRNA levels in isolated endothelial cells. Fig.11 A) from control and GRECKO Western blot analysis of GR protein levels in isolated endothelial cells from mouse kidney, and ( Fig.11 B) qPCR analysis of GR mRNA expression. n = 7 biologically independent control mice, n = 8 biologically independent GR ECKO Mice. Three independent experiments were analyzed. Data are mean ± SEM. Statistical significance was analyzed using Student's t-test (unpaired two-tailed). Significance: *p<0.05, **p<0.01, ***p<0.001.

[0034] Fig.12 Physiological characteristics of non-diabetic mice are shown. Non-diabetic control, GR ECKO 、Apoe - / - Body weight, blood glucose, kidney weight / body weight, heart weight / body weight, liver weight / body weight, albumin to creatinine ratio, plasma triglycerides, plasma cholesterol, and systolic blood pressure in DKO and DKO mice. n = 6 biologically independent mice per group, except for blood pressure measurements, which were n = 8 biologically independent mice per group, combined from 2 separate experiments. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p < 0.05, **p < 0.01, ***p < 0.001.

[0035] Fig.13 A-13B shows the assessment of glomerular fibrosis in non-diabetic and diabetic mice. Fig.13 A) Non-diabetic and diabetic controls, GR ECKO 、Apoe - / - Periodic acid-Schiff (PAS) staining in kidneys of DKO and DKO mice. Scale bar 50 μm. n = 7 biologically independent mice per group, combined from 2 separate experiments. Representative images are shown. ( Fig.13 B) Non-diabetic and diabetic controls, GR ECKO 、Apoe - / - Immunohistochemical analysis of vimentin expression in kidneys of A and DKO mice. n = 7 biologically independent mice per group, combined from 2 separate experiments. Scale bar, 50 μm. Representative images are shown; original magnification 200x.

[0036] Fig.14 The expression of Snail1, PPARα, and HIF1α in non-diabetic and diabetic mice is shown. ECKO 、Apoe - / -Immunohistochemical analysis of Snail1, PPARα, and HIF1α expression in the kidneys of DKO and DKO mice. Scale bar, 50 μm. n = 7 biologically independent mice per group, combined from 2 separate experiments. Representative images are shown; original magnification 200x.

[0037] Fig.15 Collagen I and fibronectin deposition in nondiabetic and diabetic mice are shown. ECKO 、Apoe - / - Immunofluorescence analysis of collagen I and fibronectin in the kidneys of DKO and DKO mice. FITC-labeled collagen I, rhodamine-labeled fibronectin, and DAPI were used. Scale bar, 50 μm. n=7 biologically independent mice per group. Representative images from 2 separate experiments are shown; original magnification 300x.

[0038] Fig.16 A-16C shows that loss of endothelial GR exacerbates fibrosis in a mouse model of unilateral ureteral obstruction (UUO). Fig.16 A) Schematic diagram of the UUO model. Control littermates and GR ECKO The left kidney of the mice was ligated. The kidneys were removed on days 5 and 10. Fig.16 B) Masson trichrome and Sirius red staining in the contralateral kidney and the kidney operated by UUO were analyzed. Representative images are shown; original magnification 200 times. Fibrosis area (%) and relative collagen deposition (RCD%) were measured using the ImageJ program. n = 8 biologically independent mice per group, combined from 3 independent experiments. Scale bar, 50 μm. Data are mean ± SEM. Statistical significance was calculated using two-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p<0.05. ( Fig.16 C) In control and GR ECKO Immunofluorescence analysis of collagen I, aquaporin / α-SMA, and aquaporin / fibronectin was performed in the contralateral and UUO-operated kidneys of mice. FITC-labeled collagen I, α-SMA, and fibronectin, rhodamine-labeled aquaporins, and DAPI were used. Representative images are shown; original magnification 300x. Scale bar 50 μm. n = 7 biologically independent mice per group, combined from 2 experiments. Con: control; Contra: contralateral kidney; FN: fibronectin.

[0039] Fig.17 A-17B shows dexamethasone management in urinary tract obstruction. Fig.17A) Masson trichrome staining in dexamethasone-treated and vehicle-treated UUO mice; original magnification 200x. Mice were treated with 1 mg / kg dexamethasone in drinking water until the day of sacrifice. Time-dependent analysis on days 10 and 15 is shown. Representative images are shown. Relative area of ​​fibrosis (%) was measured using the ImageJ program. n = 7 biologically independent mice per group, combined from 2 independent experiments. Scale bar 50 μm. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: ***p<0.001. ( Fig.17 B) Masson trichrome staining in dexamethasone-treated and vehicle-treated non-diabetic CD-1 mice (original magnification 200x). Representative images are shown. The relative area of ​​fibrosis (%) was measured using the ImageJ program. n = 6 biologically independent mice per group. Scale bar 50 μm. Data are mean ± SEM. Data were analyzed by Student's t-test (unpaired two-tailed).

[0040] Fig.18 Diabetic nephropathy is shown to be associated with cytokine and chemokine reprogramming. Plasma cytokines and chemokines in non-diabetic control and diabetic CD-1 and C57BL / 6 mice were measured using cytokine array analysis (Luminex). n = 5 biologically independent mice per group. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. C-CD1: non-diabetic control CD1, DM-CD1: diabetic CD-1, C-BL6: non-diabetic control C57BL / 6, DM-BL6: diabetic C57BL / 6 mice.

[0041] Fig.19 A-19B demonstrates non-diabetic and diabetic GR ECKO and cytokine and chemokine reprogramming in DKO mice. Fig.19 A) Non-diabetic and diabetic GR were measured by using cytokine array analysis (Luminex). ECKO Plasma cytokines and chemokines in DKO mice. n = 5 biologically independent mice per group. Data are mean ± SEM. Fig.19B) Relative gene expression of the indicated cytokines in diabetic kidneys was assessed. n = 6 biologically independent mice per group. 18S was used to normalize expression levels. Data are mean ± SEM. In both panels, statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p < 0.05, **p < 0.01, ***p < 0.001.

[0042] Fig. 20 A-20D shows that IL-6 neutralization rescues the fibrotic phenotype in the kidneys of diabetic CD-1 mice. Non-diabetic and diabetic CD-1 mice were injected intraperitoneally with IL-6 IgG and control IgG at a dose of 3 mg / kg three times a week for 4 weeks. Fig. 20 A-20B) Western blot analysis of vimentin and α-SMA in isolated endothelial cells from CD-1 mice stimulated with recombinant IL1β, IL-6, TNFα, and TGFβ. Representative blots from 3 independent experiments were analyzed. Densitometry calculations relative to β-actin are shown. ( Fig. 20 C) Western blot analysis of vimentin in TGFβ-stimulated and unstimulated endothelial cells treated with N-IL1β (neutralizing antibody-IL-1β) and N-IL-6 (neutralizing antibody-IL-6) antibodies. Representative blots from 3 independent experiments were evaluated. Densitometry calculations relative to β-actin are shown. ( Fig. 20 D) IL-6 neutralization in control and diabetic CD-1 mice. Representative images of Masson trichrome and Sirius red staining (original magnification 300x) are shown. n = 7 biologically independent mice per group, a combination of 2 independent experiments. Relative area of ​​fibrosis (%) and relative collagen (%) were measured using the ImageJ program. Scale bar is 50 μm. Data are mean ± SEM. For all panels, statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0043] Fig.21 Demonstrated non-diabetic GR ECKO IL-6 neutralization in nondiabetic GR and DKO mice. ECKOIL-6 IgG and control IgG were injected intraperitoneally 3 times a week in DKO and DKO mice at a dose of 3 mg / kg for 4 weeks. Representative images of Masson trichrome (top row) and Sirius red staining (bottom row) are shown (original magnification 300 times). n = 6 biologically independent mice per group, a combination of 2 experiments. The relative area (%) of fibrosis and relative collagen (%) were measured using the ImageJ program. The scale bar is 50 μm. Data are mean ± SEM. Statistical significance was analyzed using one-way ANOVA and Tukey post hoc test.

[0044] Fig. 22 A-22C shows gene expression analysis of Wnt signaling and fibrogenic markers. Fig. 22 A) Non-diabetic and diabetic controls, GR ECKO 、Apoe - / - Endothelial cells isolated from kidneys of DKO and DKO mice were analyzed for relative mRNA levels of Axin2, Tcf, α-SMA, fibronectin, Snail1, Hif1α, and Pparα determined by qRT-PCR. Endothelial cells from n = 6 biologically independent mice per group were evaluated. Combined data from 4 independent experiments were analyzed. Data are mean ± SEM. ( Fig. 22 B) The results of the study on the relationship between the expression of GR and non-diabetic and diabetic controls ECKO Endothelial cells isolated from cultured glomeruli of mouse kidney were analyzed for relative mRNA levels of Axin2, Tcf, and fibronectin as determined by qRT-PCR. Endothelial cells from n=6 biologically independent mice per group were analyzed. Combined data from 3 independent experiments were analyzed. Data are mean ± SEM. Fig. 22 A) and ( Fig. 22 In B), statistical significance was analyzed using one-way ANOVA and Tukey post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. ( Fig. 22 C) Immunohistochemical analysis of β-catenin in kidneys of the indicated groups of mice. Representative images are shown; original magnification is 300x. n = 7 biologically independent mice per group, combined from 3 independent experiments. Scale bar, 50 μm.

[0045] Fig.23 A-23C exhibits endothelial cell characteristics. Fig.23 A) In non-diabetic and diabetic controls, GR ECKO 、Apoe - / -Endothelial cell leakage was assessed 2 h after fluorescein isothiocyanate (FITC)-dextran injection in the kidneys of DKO and DKO mice. FITC-dextran, rhodamine-labeled CD31, and DAPI were used. Merged images and representative figures are shown (original magnification 300x). Arrows indicate cells labeled only by FITC-dextran and areas of leakage. n = 7 biologically independent mice per group, combined from 3 experiments. Scale bar 50 μm. Quantification is shown below the figure. ND: non-diabetic, D: diabetic. Data are mean ± SEM. ( Fig.23 B) The permeability of isolated endothelial cell monolayers was assessed using Transwell culture inserts by measuring the transfer of FITC-dextran added to the upper compartment to the lower compartment. After 24 hours, the FITC-dextran concentration in the lower compartment was measured using a fluorescent plate reader. Endothelial cells isolated from 6 biologically independent mice were assessed. Three independent experiments were analyzed. ( Fig.23 C) Through Fig.23 Endothelial cell density (%) measured by CD31 staining of A. Endothelial cells from 6 biologically independent mice were evaluated. Three independent experiments were analyzed. Data are mean ± SEM. For all groups, statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0046] Fig.24 A-24J showed that inhibition of Wnt signaling abolished the fibrogenic phenotype in mice. Fig.24 A) Immunohistochemical analysis of β-catenin expression in kidneys of control and diabetic CD-1 and C57BL / 6 mice. n = 8 biologically independent mice per group were evaluated, combined from 2 independent experiments. Scale bar, 50 μm. Representative images are shown; original magnification is 200x. ( Fig.24 B) Relative mRNA levels of Axin2, Tcf, and fibronectin determined by qRT-PCR were analyzed in kidneys of control and diabetic CD-1 and C57BL / 6 mice. n=6 biologically independent mice per group were evaluated, combined from 2 experiments. Expression was normalized to the internal control 18S. ( Fig.24 C) Schematic diagram of the treatment regimen of Wnt inhibitor (LGK974; 5 mg / kg body weight) in diabetic CD-1 mice, and ( Fig.24 D) Schematic representation of the treatment regimen in UUO mice. Fig.24E) Masson trichrome and Sirius red staining in kidneys of diabetic CD-1 mice treated with diabetes and Wnt inhibitors. Representative images are shown (original magnification 300x). Relative area of ​​fibrosis (%) and relative collagen deposition (%) were measured using the ImageJ program. n = 8 biologically independent mice per group were evaluated, combined from 2 experiments. Scale bar 50 μm. ( Fig.24 F) Masson trichrome and Sirius red staining in kidneys of UUO and Wnt inhibitor treated UUO mice. Representative images are shown (original magnification 300x). Relative area of ​​fibrosis (%) and relative collagen deposition (%) were measured using the ImageJ program. n = 8 biologically independent mice per group were evaluated, from a combination of 2 experiments. Scale bar 50 μm. ( Fig.24 G-24H) GR protein levels in CD31-positive cells were analyzed by immunofluorescence in the 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 300x. n = 7 biologically independent mice per group were evaluated, combined from 4 experiments. Scale bar, 50 μm. ( Fig.24 I-24J) Immunohistochemical analysis of active-catenin expression in Wnti-treated diabetic mice and Wnti-treated UUO mice. n = 7 biologically independent mice per group were evaluated, from a combination of 3 experiments. Scale bar 30 μm. Data are mean ± SEM. For all groups, statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0047] Fig.25 It was shown that inhibition of Wnt signaling in diabetic mice disrupts cytokine and chemokine reprogramming. Plasma cytokines and chemokines in diabetic CD-1 mice treated with and without Wnt were measured using cytokine array analysis (Luminex). n = 5 biologically independent mice per group. Data are mean ± SEM. Statistical significance was analyzed using Student's t-test (unpaired two-tailed). Significance: *p<0.05.

[0048] Fig.26 Non-diabetic controls and GR ECKOPhysiological characteristics of Wnt inhibitor treatment in mice. Physiological parameters including body weight, blood glucose and kidney weight / body weight are shown. n=7 biologically independent mice per group were analyzed, combined from 2 experiments. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test.

[0049] Fig. 27 A-27B showed a significant difference in the expression of Wnt inhibitor-treated non-diabetic controls and GR ECKO Histological analysis of mouse kidneys. Fig. 27 A) Non-diabetic controls and GR treated with Wnt inhibitors ECKO Masson trichrome staining (MTS) and Sirius red staining in 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 per group were analyzed. Data are mean ± SEM. Scale bar, 50 μm. ( Fig. 27 B) Non-diabetic controls and GR treated with Wnt inhibitors ECKO Immunohistochemical analysis of β-catenin expression in the kidney of mice. Scale bar, 30 μm. n = 6 biologically independent mice per group were analyzed, combined from 2 independent experiments. Representative images are shown; original magnification 300x. Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0050] Fig.28 The expression of Snail1, PPARα, and HIF1α in non-diabetic and diabetic mice treated with Wnt inhibitors is shown. ECKO The expression of Snail1, PPAR, and HIF1 in mice was analyzed immunohistochemically. Scale bar, 50 μm. n = 7 biologically independent mice per group were analyzed, combined from 3 independent experiments. Representative images are shown; original magnification 200X.

[0051] Fig.29Snail1, PPARα, active β-catenin, and HIF1α in isolated cells from Wnt inhibitor-treated diabetic mice are shown. Western blot analysis of Snail1, PPARα, active β-catenin, and HIF1α proteins in isolated endothelial cells was performed. Representative blots are shown. Densitometry calculations are from 3 independent experiments combined. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0052] Fig.30 EndMT and EMT analysis in the kidneys of Wnt inhibitor-treated mice is shown. Immunofluorescence analysis of α-SMA / CD31 / DAPI and E-cadherin / α-SMA / DAPI in the kidneys of Wnt inhibitor-treated non-diabetic and diabetic mice was performed. Upper panel: FITC-labeled α-SMA, rhodamine-labeled CD31, and DAPI in blue; lower panel: FITC-labeled E-cadherin in green, rhodamine-labeled α-SMA in red, and DAPI in blue. Representative images are shown; original magnification is 400x. Scale bar, 50 μm. n = 6 biologically independent mice per group were analyzed, combined from 3 independent experiments. NC: non-diabetic control; DC: diabetic control; D: diabetic.

[0053] Fig.31 A-31B shows fatty acid uptake and fatty acid oxidation in isolated endothelial cells. Fig.31 A) Fatty acid uptake via [ 14 C] palmitic acid uptake. Fig.31 B) fatty acid oxidation to [ 14 CO2] release. Use [ 14 C] Palmitic acid was used as substrate and CPM counts were normalized to protein. In both cases, endothelial cells isolated from n=5 biologically independent mice per group were analyzed in triplicate. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001.

[0054] Fig.32 Gene expression of fatty acid transporters in nondiabetic and diabetic mice is shown. ECKOEndothelial cells isolated from the kidneys of mice were analyzed for relative mRNA levels of Fatp1, Fatp4, and Cd36 determined by qRT-PCR. Endothelial cells isolated from n=5 biologically independent mice per group were analyzed in triplicate. Data are mean ± SEM. Statistical significance was analyzed using one-way ANOVA and Tukey post hoc test. Significance: *p<0.05.

[0055] Fig.33 A-33F demonstrates that endothelial GR is essential for the effects of antidyslipidemic drugs in diabetic nephropathy. Fig.33 A) Masson trichrome (MTS) and Sirius red staining in kidneys of control, diabetic mice, diabetic mice treated with fenofibrate, etomoxir, C75, and simvastatin. Representative images are shown (original magnification 300x). Relative area of ​​fibrosis (%) and relative collagen deposition (%) were measured using the ImageJ program. n = 6 biologically independent mice per group were analyzed, from a combination of 2 experiments. Scale bar is 50 μm. ( Fig.33 B) GR / CD31 co-immunolabeling was analyzed by fluorescence microscopy. FITC-labeled GR, rhodamine-labeled CD31, and DAPI were used. Representative images are shown; original magnification 300x. Scale bar 50 μm. n = 6 biologically independent mice per group were analyzed, combined from 2 experiments. ( Fig.33 C) PCR gene analysis of fibronectin and α-SMA in kidney. 18S was used as internal control. n = 6 biologically independent mice per group. ( Fig.33 D) Blood glucose measurements in control, diabetic, diabetic mice treated with fenofibrate, etomoxir, C75, and simvastatin. n = 6 biologically independent mice per group. Fig.33 E) measured radiolabeled [ 14 C] Palmitic acid oxidation and [ 14 CO2] release. CPM counts were performed for each sample. Endothelial cells isolated from 6 biologically independent mice were evaluated. Three independent experiments were analyzed. Data are mean ± SEM. ( Fig.33F) Immunohistochemical analysis of CPT1a and β-catenin in kidneys of control, diabetic, diabetic mice treated with fenofibrate, C75, etomoxir, and simvastatin. n = 6 biologically independent mice per group. Representative images are shown; original magnification 400x. Scale bar 50 μm. Data are mean ± SEM. For all groups, statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p<0.05, **p<0.01, ***p<0.001. Con: control, DM: diabetes, feno: fenofibrate.

[0056] Fig.34 Physiological characteristics of non-diabetic mice treated with etomoxir and C75 are shown. Body weight, blood glucose and kidney weight / body weight ratio are shown as well as [ 14 CO2] release represents fatty acid oxidation. 14 C] Palmitic acid was used as substrate and CPM counts were normalized to protein. n = 6 biologically independent mice per group. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: *p < 0.05.

[0057] Fig.35 Shown is the histological analysis of kidneys from non-diabetic mice treated with etomoxir and C75. ECKO Masson trichrome staining (upper panel) and Sirius red (lower panel) in mice. ECKO Etomoxir (20 mg / kg) and C75 (15 mg / kg) were administered 3 times per week (IP) for 3 weeks in control littermates. Representative images are shown; original magnification 400x. Fibrosis area (%) and relative collagen deposition (%) in n=6 biologically independent mice per group combined from 2 independent experiments. Scale bar 50 μm. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: **p<0.01.

[0058] Fig.36GR overexpression inhibits fibrogenic markers in vitro. Western blotting and corresponding quantitative analysis of protein levels of GR and collagen I (normalized to β-actin) and β-catenin and α-SMA (normalized to HSP90) in HUVECs treated with empty vector, GR overexpression vector, empty vector + TGFβ2, or GR overexpression vector + TGFβ2. Representative blots are shown. Data from 3 independent experiments were combined for analysis. Data are mean ± SEM. Statistical significance was calculated using one-way ANOVA and Tukey's multiple comparison post hoc test. Significance: ***p<0.001.

[0059] Fig.37 We show that EC-specific inhibition of canonical Wnts leads to significant improvements in renal and cardiac fibrosis and provides evidence for the involvement of the Wnt canonical pathway. These data were generated using the endothelial-specific LRP5 / 6KO mouse model, a novel mouse model that inhibits canonical Wnt signaling in the LRP5 / 6 receptor.

[0060] Fig.38 A-38H demonstrated that pharmacological inhibition of Wnt signaling abolished the fibrogenic phenotype in db / db mice. Fig.38 A) Postprandial blood glucose measurement in Wnt inhibitor treated db / db mice and untreated control db / db mice. LGK-974 (Wnt inhibitor) was treated at a dose of 5 mg / kg body weight by oral gavage. Fig.38 B) GTT analysis in Wnt inhibitor treated db / db mice and untreated control db / db mice. N=6 mice were analyzed per group. Fig.38 C) Area under the curve (AUC) of Wnt inhibitor treated db / db mice and untreated control db / db mice. N = 6 mice were analyzed per group. ( Fig.38 D) Physiological parameters such as body weight, epididymal fat, kidney weight, liver weight, and heart weight of Wnt inhibitor-treated db / db mice and untreated control db / db mice. N = 6 mice were analyzed for each group. Fig.38 E) MTS and Sirius red staining analysis in Wnt inhibitor treated db / db mice and untreated control db / db mice. Representative images at 30× are shown. N=6 mice were analyzed per group. Relative area of ​​fibrosis (RAF) and relative collagen deposition (RCD) were quantified using Image J software program. Fig.38 F) MTS and Sirius red staining analysis in cardiac sections of Wnt inhibitor-treated db / db mice and untreated control db / db mice. Representative images at 20× are shown. N=6 mice were analyzed per group. ( Fig.38G) MTS in liver sections of Wnt inhibitor-treated db / db mice and untreated control db / db mice. Fig.38 H) MTS in adipose tissue sections. Representative images at 20× are shown. N = 6 animals were analyzed per group. Scale bar, 50 μm. All data represent mean ± SEM. Statistical significance was analyzed using unpaired t-test. *P ≤ 0.05. DETAILED DESCRIPTION OF THE INVENTION

[0061] Unless defined otherwise, 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.

[0062] 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 employed in its sense including "and / or" unless the context clearly dictates otherwise.

[0063] The term "antibody" refers to all immunoglobulin isotypes (e.g., IgG, IgA, IgE, IgM, IgD, and IgY), including various monomeric, polymeric, and chimeric forms, unless otherwise indicated. Specifically included in the term "antibody" are 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.

[0064] The term "antigen binding fragment" refers to any proteinaceous structure that may exhibit binding affinity for a specific antigen. Antigen binding fragments include fragments produced by any known technology, such as enzymatic cleavage, peptide synthesis and recombinant technology. Some antigen binding fragments are composed of parts of intact antibodies, and the parts retain the antigen binding specificity of the parent antibody molecule. For example, an antigen binding fragment may include at least one variable region (whether a heavy chain or a light chain variable region) or one or more complementary determining regions (CDRs) of an antibody known to bind to a specific antigen. Examples of suitable antigen binding fragments include, but are not limited to, single-chain molecules such as Fab, F(ab')2, Fc, Fabc, Fv molecules, scFv and disulfide-linked Fv (sdFv), intrabody, diabody, minibody, linear antibody, single domain antibody such as sdAb (VL or VH), camel nanobody (VHH domain), multispecific antibodies formed by antibody fragments, single antibody light chain, single antibody heavy chain, antibody chain or Chimeric fusions between CDR and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy chain and one light chain, monovalent fragments consisting of VL, VH, CL and CH1 domains, or monovalent antibodies as described in WO2007059782 (the entire contents of which are incorporated herein by reference), bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region, Fd fragments consisting essentially of VH and CH1 domains, dAb fragments, or isolated CDRs, etc. All antibody isotypes can be used to produce antigen-binding fragments. In addition, antigen-binding fragments may include non-antibody protein frameworks that may successfully incorporate polypeptide segments, such as protein scaffolds, in an orientation that confers affinity for a given target antigen. 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 mentioned in the phrase.

[0065] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen binding site (called a paratope) in the variable region of an antibody molecule. A single antigen may have more than one epitope. Therefore, different antibodies may bind to different regions on the antigen and may 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 the antigen bound by an antibody. An epitope can be defined as structural or functional. A functional epitope is typically a subset of a structural epitope and has those residues that directly contribute to the affinity of the interaction. An epitope may also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope may include a determinant, which is a chemically active surface group of a molecule such as an amino acid, a sugar side chain, a phosphate group, or a sulfonyl group, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features.

[0066] 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, cattle, horses, sheep, pigs, etc.) and experimental animal models (e.g., mice, rabbits, rats). Animals include all vertebrates, such as mammals and non-mammals, such as mice, sheep, dogs, cattle, birds, ducks, geese, pigs, chickens, amphibians and reptiles. In preferred embodiments, the subject is a human. In some embodiments, the subject is an individual in need of prevention or treatment of dyslipidemia or related disorders or conditions.

[0067] The terms "treat" or "treatment" of a state, disorder, or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance and development of at least one clinical or subclinical symptom of a state, disorder, or condition in a subject who may already have or be susceptible to the state, disorder, or condition but does not yet experience or exhibit clinical or subclinical symptoms of the state, disorder, or condition; or (2) inhibiting the state, disorder, or condition, i.e., arresting, reducing, or delaying the disease or its recurrence (in the case of maintenance therapy) or the development of at least one clinical or subclinical symptom thereof; or (3) ameliorating the disease, i.e., causing regression of the state, disorder, or condition or at least one clinical or subclinical symptom thereof. The benefit to the subject being treated is statistically significant or at least perceptible to the patient or physician.

[0068] The term "need for treatment" as used herein refers to a judgment made by a physician or other caregiver that a subject needs treatment or will benefit from treatment. This judgment is made based on various factors within the physician's or caregiver's professional scope.

[0069] The terms "therapeutically effective amount" and "effective amount" are used interchangeably herein and refer to an amount of an agent, alone or as part of a pharmaceutical composition, administered to a subject in a single dose or as part of a series of doses, which, when administered to a subject, produces any detectable positive effect on any symptom, aspect or feature of a disease, disorder or condition. A therapeutically effective amount can be determined by measuring relevant physiological effects and can be adjusted with respect to dosing regimens and diagnostic analysis of the subject's condition, etc.

[0070] The term "pharmaceutically acceptable" as used herein refers to molecular entities and other ingredients in such compositions that are physiologically tolerable and generally do not produce adverse reactions when administered to mammals (e.g., humans). Preferably, the term "pharmaceutically acceptable" refers to approved by a regulatory agency of the U.S. federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, and can be used in mammals, particularly humans.

[0071] The term "carrier" or "pharmaceutically acceptable carrier" as used herein refers to any clinically useful solvent, diluent, adjuvant, excipient, receptor, vehicle, etc., used to prepare a mixture for a pharmaceutical composition.

[0072] The term "about" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within a standard deviation that is acceptable according to the conventions in the art. Alternatively, "about" can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and still 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 of a value, preferably within a factor of 2. When describing specific values ​​in the application and claims, the term "about" is implicit unless otherwise stated, in which case it means within an acceptable error range for the specific value.

[0073] As used in this specification and claims, the words "comprise" (and any form of inclusion, such as "contains" and "includes"), "have" (and any form of having, such as "have" and "have"), "include" (and any form of inclusion, such as "includes" and "covers"), or "contains" (and any form of containing, such as "includes" and "including") are inclusive or open-ended and do not exclude additional unrecited elements or process steps.

[0074] In light of the present disclosure, conventional molecular biology, microbiology, and recombinant DNA techniques known to those skilled in the art may be employed. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 (hereinafter "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Vols. I and II (Glover, ed. 1985); Oligonucleotide Synthesis (Gait, ed. 1984); Nucleic Acid Hybridization (Hames and Higgins, ed. 1985); Transcription And Translation (Hames and Higgins, ed. 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, 1986. Sons, Inc. 1994; etc.

[0075] In one embodiment of the present disclosure, there is provided a method of 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.

[0076] Conditions or diseases that may be treated by the methods of the present disclosure include, but are not limited to, organ fibrosis, renal disease, dyslipidemia, hypertension, hyperlipidemia, hypercholesterolemia, cardiovascular disease, peripheral arterial disease, atherosclerosis, coronary artery disease, coronary heart disease, and stroke.

[0077] In some embodiments, the organ fibrosis is renal fibrosis.

[0078] In some embodiments, the kidney disease is diabetic nephropathy.

[0079] In some embodiments, the method is effective in preventing or treating nephropathy. In some embodiments, the method is effective in reducing fibrosis in the kidneys of a subject. In some embodiments, the method is effective in reducing collagen deposition in the kidneys of a subject. In some embodiments, the method is effective in reducing the accumulation of collagen in the kidneys of a subject. In some embodiments, the method is effective in reducing glomeruloscerosis in a subject.

[0080] The term "dyslipidemia" refers to abnormal levels of lipoproteins in plasma, including decreased and / or increased levels of lipoproteins (eg, increased levels of LDL and / or VLDL, and decreased levels of HDL).

[0081] As used herein, the term "hypercholesterolemia" refers to a condition in which cholesterol levels are elevated beyond a desired level. In some embodiments, this means that serum cholesterol levels are elevated. In some embodiments, the desired level takes into account a variety of "risk factors" known to those skilled in the art (and described or mentioned herein). "Familial hypercholesterolemia" refers to hypercholesterolemia caused by a genetic mutation located on chromosome 19.

[0082] 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 the early and overt stages of diabetic nephropathy, whether or not diagnosed, although diabetic nephropathy is most typically diagnosed by a clinician or physician.

[0083] The term "atherosclerosis" as used herein refers to an arterial disease characterized by narrowing of the arteries due to the accumulation of plaque within the arteries. The term "atherosclerosis-related conditions" refers to atherosclerotic cardiovascular disease (ASCVD) and other such chronic inflammatory diseases driven by cholesterol deposition. Atherosclerosis-related conditions include, but are not limited to: ASCVD, coronary heart disease, such as myocardial infarction, angina pectoris, and coronary artery stenosis; cerebrovascular disease, such as transient ischemic attack, ischemic stroke, and carotid stenosis; peripheral arterial disease, such as claudication; aortic atherosclerotic disease, such as abdominal aortic aneurysm and descending thoracic artery aneurysm; hypertension; peripheral vascular disease; coronary artery disease; aortic aneurysm; carotid artery disease; coronary atherosclerosis; heart attack; acute coronary syndrome and stroke.

[0084] As used herein, the term "coronary heart disease / coronary artery disease" refers to the narrowing of the small blood vessels that supply blood and oxygen to the heart, often as a result of atherosclerosis.

[0085] In various embodiments of the methods described herein, the subject is a human.

[0086] In some embodiments, the IL-6 activity inhibitor is an antibody or antigen-binding fragment.

[0087] In some embodiments, the inhibitor is an IL-6 neutralizing antibody or an antigen-binding fragment thereof. In some embodiments, the inhibitor is sirukumab, siltuximab, or olozumab, or an antigen-binding fragment thereof.

[0088] 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 sarelumab or an antigen-binding fragment thereof.

[0089] In some embodiments, the antibodies or antigen-binding fragments described herein specifically bind to IL-6 or IL-6 receptor with high affinity, e.g., K D Less than about 1×10 -8 M, for example but not limited to, about 1-9.9 (or any range or value therein, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9)×10 -9 M,10 -10 M,10 -11 M,10 -12 M,10 -13 M,10 -14 M,10 -15 M or any range or value therein, as determined by, for example, biofilm interferometry, surface plasmon resonance, or Kinexa methods, as practiced by one skilled in the art. An example affinity is equal to or less than 1×10 -10 M. Another example affinity is equal to about 3.8×10 -11 M.

[0090] Methods for testing the ability of an antibody to bind to a target peptide or any portion thereof are known in the art and include any antibody-antigen binding assay, e.g., biofilm interferometry, radioimmunoassay (RIA), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assays. In some embodiments, the K D Values ​​were determined using biofilm layer interferometry.

[0091] 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.

[0092] 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 receptors can also be used in the methods of the present disclosure. The terms "competition" or "cross-competition" as used herein refer to an antibody or antigen-binding fragment thereof binding to an antigen and inhibiting or blocking the binding of another antibody or antigen-binding fragment thereof. The term also includes competition between two antibodies in two directions (wherein the first antibody binds and blocks the binding of the second antibody, and vice versa). In some embodiments, the competing antibody and the antibody described herein may bind to the same epitope. Alternatively, the competing antibody and the antibody described herein may bind to different but overlapping epitopes, so that the binding of one antibody inhibits or blocks the binding of the second antibody, for example, by steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, for example, by real-time label-free biofilm interferometry. Cross-competition between two antibodies can be expressed as the binding of the second antibody less than the background signal caused by self-binding (wherein 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-background binding (wherein the first and second antibodies are the same antibody).

[0093] Antibodies or antigen-binding fragments thereof that bind to the same epitope as the antibodies or antigen-binding fragments described herein can also be used in the methods of the present disclosure.

[0094] The antibodies or antigen-binding fragments described herein can be any of various antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. In some embodiments, the antibody isotype is an 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 largely determined by the amino acid sequence and arrangement of the CDR. Therefore, the CDR of one isotype can be transferred to another isotype without changing the antigen specificity. Alternatively, a technique has been established that allows a hybridoma to be converted from producing an antibody isotype to another (isotype conversion) without changing the antigen specificity. Therefore, such an antibody isotype is within the scope of the antibody or antigen-binding fragment.

[0095] In some embodiments, the IL-6 activity inhibitor provides trans signaling blockade. In one embodiment, the inhibitor is Olazicept (FE999301) or a fragment or variant thereof.

[0096] In some embodiments, the IL-6 activity inhibitor is an inhibitor of intracellular signaling. In some embodiments, the inhibitor is tofacitinib or CpG-stat3 siRNA.

[0097] The polypeptide compounds (eg, antibodies or antigen-binding fragments) described herein include variants having single or multiple amino acid substitutions, deletions or additions but retaining the biological properties (eg, binding affinity or immune effector activity) of the polypeptide compound.

[0098] These variants may include: (i) variants in which one or more amino acid residues are substituted 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 substitution group, and (iv) variants in which the antibody or antigen-binding fragment is fused or conjugated to another peptide or polypeptide (e.g., a fusion partner, a protein tag) or other chemical moiety, which may confer useful properties to the antibody or antigen-binding fragment, such as, for example, an epitope of an antibody, a polyhistidine sequence, a biotin moiety, etc. The polypeptide compounds described herein may include variants in which an amino acid residue of one species is replaced with a corresponding residue in another species (whether in a conservative or non-conservative position). In other embodiments, amino acid residues at non-conservative positions are substituted with conservative or non-conservative residues. Amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc.

[0099] Amino acid substitutions can be conservative, meaning that the substituted amino acid has similar chemical properties as the original amino acid. A skilled artisan will understand which amino acids have similar chemical properties. For example, the following groups of amino acids have 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).

[0100] Thus, embodiments of polypeptide compounds may 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.

[0101] In some embodiments of the methods provided herein, the method further comprises administering to the subject one or more other effective treatments of the intended condition or disease. Such agents may include, but are not limited to, cholesterol-lowering drugs (e.g., statins, fibrates, inhibitors of proprotein convertase subtilisin / kexin9 type), blood pressure-lowering treatments (e.g., angiotensin converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARB)), anti-inflammatory agents, antithrombotic agents, anticoagulants, inhibitors of the renin-angiotensin-aldosterone system (RAAS inhibitors), beta-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 co-transporter 2 (SGLT2) inhibitors) and / or other non-pharmacological treatments. When using combination therapy, the IL-6 inhibitor (one or more) and one or more additional agents can be present in a single composition or in multiple compositions, and the treatment can be administered simultaneously, sequentially or by some other regimen. Combination therapy can have additive or synergistic effects.

[0102] Other suitable agents that may be used in combination with the IL-6 inhibitors described herein include, but are not limited to, those Wnt inhibitors described in US Pat. Nos. 11,369,609 and 9,045,416, both of which are incorporated herein by reference in their entirety.

[0103] For example, a Wnt inhibitor to be used in combination with an IL-6 inhibitor described herein may comprise a compound having a structure according to formula (I): wherein 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 them is N, and the others are selected from N and CR; X 11 , X 12 , X 13 and X 14 One of them is N, and the others are selected from N and CR, and R, at each occurrence, is independently selected from hydrogen, halogen, cyano, methyl, difluoromethyl and trifluoromethyl, or a pharmaceutically acceptable salt thereof.

[0104] In some embodiments of the above compounds, one of X3, X4, X5 and X6 is N, and the others are CR. 10One of X is N and the other is CR. 11 , X 12 , X 13 and X 14 In some embodiments, two of them are N and the others are CR. In some embodiments, X1 is CR and R is methyl, and / or wherein X5 is CR and R is methyl. In some embodiments, one or more of the following occurs: X2 is CH, X4 is CH, X6 is CH, X8 is CH, X9 is CH, X 10 For CH, X 12 is CH, and X 13 For CH.

[0105] In specific embodiments, the compound is a compound having a structure according to Formula (I), wherein X1 is CR, wherein R is methyl; X2 is CH; X3 is N; X5 is CR, wherein R is methyl; X4 and X6 are each CH; X7 is N; X8, X9 and X 10 Each is CH; X 11 and X 14 Each is N; X 12 and X 13 Each is CH.

[0106] In a specific embodiment, the Wnt inhibitor used in combination with the IL-6 inhibitor described herein may comprise the compound LGK974 having the following structure: or a pharmaceutically acceptable salt thereof.

[0107] LGK974 can inhibit PORCN, which, without wishing to be bound by theory, is believed to be required for palmitoylation of Wnt ligands. 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 phosphorylation of LRP5 and LPR6.

[0108] The chemical structures herein are drawn according to conventional standards known in the art. Thus, when an atom (e.g., a carbon atom) is drawn with an apparent unsatisfied valence, the valence is assumed to be satisfied by a hydrogen atom, even though the hydrogen atom is not necessarily drawn explicitly. The hydrogen atom should be inferred to be part of the compound.

[0109] As will be understood by those of ordinary skill in the art, the description of a compound is intended to include salts, solvates, oxides and inclusion complexes of the compound, as well as any stereoisomers, or any such forms of the compound in any proportion of mixtures. The compounds described herein include, but are not limited to, their optical isomers, racemates, and other mixtures thereof. In these cases, a single enantiomer or diastereomer, i.e., an optically active form, can be obtained by asymmetric synthesis or the resolution of a racemate. The resolution of a racemate can be accomplished by, for example, conventional methods such as crystallization in the presence of a resolving agent, or using, for example, a chiral high pressure liquid chromatography (HPLC) column chromatography. In addition, such compounds include Z- and E-type forms (or cis and trans forms) of compounds with carbon-carbon double bonds. When the compounds described herein exist in a variety of tautomeric forms, the term "compound" is intended to include all tautomeric forms of the compound. Such compounds also include crystalline forms, including polymorphs and inclusion compounds. Similarly, the term "salt" is intended to include all tautomeric forms and crystalline forms of the compound.

[0110] Therefore, according to some embodiments of the present disclosure, the compounds as described herein, included in the context of pharmaceutical compositions and methods of treatment, are provided in the form of salts. A "pharmaceutically acceptable salt" of a compound refers to a salt of a pharmaceutically acceptable compound. It is desirable to retain or improve the biological effectiveness and properties of the free acid and base of the parent compound as defined herein, or to utilize compound salts of basic, acidic or charged functional groups inherent in the molecule, and these compound salts are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts are also described in, for example, Berge et al., "Pharmaceutical Salts", J. Pharm. Sci. 66, 1-19 (1977). Non-limiting examples of such salts include: (1) acid addition salts, which are formed at basic or positively charged functional groups by the addition of inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, carbonate formers, and the like; or with organic acids such as acetic acid, propionic acid, lactic acid, oxalic acid, glycolic acid, pivalic acid, tert-butylacetic acid, β-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, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethylsulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, cyclohexylaminosulfonic acid, benzenesulfonic acid, p-aminobenzenesulfonic acid (sulfanilic acid) acid), 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 3-phenylpropionic acid, dodecylsulfonic acid, dodecylsulfonic acid, oleic acid, palmitic acid, stearic acid, lauric acid, pamoic acid, palmitic acid, pantothenic acid, lactobionic acid, alginic acid, galactaric acid, galacturonic acid, gluconic acid, glucoheptonic acid, glutamic acid, naphthoic acid, hydroxynaphthoic acid, salicylic acid, ascorbic acid, stearic acid, muconic acid (2) base addition salts, which are formed when the acidic protons present in the parent compound are replaced by metal ions, including alkali metal ions (e.g., lithium, sodium, potassium), alkaline earth metal ions (e.g., magnesium, calcium, barium) or other metal ions such as aluminum, zinc, iron, etc.; or coordinated with organic bases (e.g., ammonia, ethylamine, diethylamine, ethylenediamine, N,N'-dibenzylethylenediamine, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, piperazine, chloroprocaine, procaine, choline, lysine, etc.).

[0111] Pharmaceutically acceptable salts can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Typically, such salts are prepared by reacting the free acid or base form of the compound with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or in a mixture of the two. The salt can be prepared in situ during the final isolation or purification of the compound, or by reacting the compound in free acid or free base form with the desired corresponding base or acid separately and isolating the salt thus formed. The term "pharmaceutically acceptable salt" also includes zwitterionic compounds containing cationic groups covalently bonded to anionic groups because they are "inner salts". It should be understood that all acids, salts, bases and other ionic and non-ionic forms of the compounds described herein are intended to be included. For example, if a compound is shown as an acid herein, the salt form of the compound is also included. Similarly, if a compound is shown as a salt, the acid and / or base form is also included.

[0112] The pharmaceutical compositions of the present disclosure may include compounds as described herein and pharmaceutically acceptable carriers or excipients. Pharmaceutically acceptable carriers may include physiologically acceptable compounds that act, for example, 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, low molecular weight proteins, compositions that reduce the removal or hydrolysis of glycopeptides, or excipients or other stabilizers and / or buffers. Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known, including, for example, phenol and ascorbic acid. Detergents may also be used to stabilize or increase or decrease the absorption of the pharmaceutical composition, including liposome carriers. Pharmaceutically acceptable carriers and formulations are known to technicians and are described in detail in scientific and patent literature, see, for example, Remington's Pharmaceutical Science latest edition, Mack Publishing Company, Easton, Pa. ("Remington's"). Those skilled in the art will appreciate that the choice of a pharmaceutically acceptable carrier (including a physiologically acceptable compound) depends, for example, on the route of administration of the composition and its specific physicochemical properties.

[0113] The composition can be administered by any suitable means, for example, orally, such as in the form of a pill, tablet, capsule, granule or powder; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular, peritoneal or intrasternal injection or using an infusion technique (e.g., as a sterile injectable aqueous or non-aqueous solution or suspension); nasally, such as by inhalation spray, aerosol, mist or spray; topically, such as in the form of a cream, ointment, salves, powder or gel; transdermally, such as in the form of a patch; transmucosal; or rectally, such as in the form of a suppository. The compositions of the present disclosure can also be administered in a form suitable for immediate release or extended release. Immediate release or extended release can be achieved by using a suitable pharmaceutical composition, or, particularly in the case of extended release, by using a device such as a subcutaneous implant or an osmotic pump.

[0114] In various embodiments, the pharmaceutical composition is formulated for oral administration. Suitable forms for oral administration include, but are not limited to, tablets, capsules, tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups, solutions, microbeads, or elixirs. Pharmaceutical compositions intended for oral administration may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may include one or more preparations, such as sweeteners, flavoring agents, colorants, and preservatives, to provide pharmaceutically acceptable preparations. Tablets, capsules, etc. typically contain active ingredients mixed with non-toxic pharmaceutically acceptable carriers or excipients, which are suitable for the manufacture of tablets. These carriers or excipients may include, for example, diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granules and disintegrants, such as corn starch or alginic acid; binders, such as starch, gelatin, or gum arabic, and lubricants, such as magnesium stearate, stearic acid, or talc.

[0115] Tablets, capsules etc. suitable for oral administration can be uncoated, and known techniques can also be used for coating, to delay decomposition and absorption in the gastrointestinal tract, thereby providing sustained action.For example, time delay materials can be used, such as glyceryl monostearate or glyceryl distearate.They can also be coated to form permeability therapeutic tablets for controlled release by techniques known in the art.Other medicaments include biodegradable or biocompatible particles or polymeric substances, such as polyester, polyamino acid, hydrogel, polyvinyl pyrrolidone, polyanhydride, polyethylene glycol acid, ethylene-vinyl acetate, methylcellulose, carboxymethyl cellulose, protamine sulfate or lactide / glycolide copolymer, polylactide / glycolide copolymer, or ethylene-vinyl acetate copolymer, to control the delivery of the composition used.For example, oral medicaments can be wrapped in microcapsules prepared by coacervation technology or interfacial polymerization, using hydroxymethyl cellulose or gelatin-microcapsules or polymethyl methacrylate microcapsules respectively, or in colloidal drug delivery systems. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, microbeads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. The preparation of the above formulations will be apparent to those skilled in the art.

[0116] Oral preparations can also be presented 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 in admixture with excipients suitable for their preparation. This excipient can be a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinyl pyrrolidone, tragacanth and gum arabic; a dispersant or wetting agent, such as naturally occurring phosphatide (e.g., lecithin), or the condensation product of alkylene oxide and fatty acid (e.g., polyethylene glycol stearate), or the condensation product of ethylene oxide and long-chain fatty alcohol (e.g., 17ethoxy hexadecanol), or the condensation product of ethylene oxide and the partial ester derived from fatty acid and hexitol (e.g., polyoxyethylene sorbitol monooleate), or the condensation product of ethylene oxide and the partial ester derived from fatty acid and hexitol anhydride (e.g., polyethylene sorbitan monooleate). Aqueous suspensions can also include one or more preservatives.

[0117] Other formulations suitable for oral use include oily suspensions. Oily suspensions can be prepared by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or a mineral oil (e.g., liquid paraffin). Oily suspensions may contain thickeners, such as beeswax, hard paraffin, or hexadecanol. Sweeteners and flavorings as described above may be added to provide a palatable oral preparation.

[0118] Dispersible powders and granules suitable for preparation of an aqueous suspension may be prepared by the addition of water to 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.

[0119] The pharmaceutical composition of the present disclosure can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil (e.g., olive oil or peanut oil), or a mineral oil (e.g., liquid paraffin), or a mixture of these. Suitable emulsifiers can be naturally occurring gums, such as gum arabic or tragacanth; naturally occurring phospholipids, such as soybeans, lecithin, and esters or partial esters derived from fatty acids; hexitol anhydrides, such as sorbitan monooleate; and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.

[0120] The pharmaceutical compositions of the present disclosure can be formulated into useful dosage units for administration by various routes, including topical, oral, subcutaneous, intravenous, and intranasal administration, among others.

[0121] Pharmaceutical compositions of the present disclosure may also include other bioactive substances in combination with compounds of the present disclosure. Such other bioactive substances may also be prepared as separate compositions and may be administered simultaneously or sequentially with compounds of the present disclosure. Non-limiting examples of available bioactive substances include statins, nicotinic acid, bile acid resins, fibric acid derivatives, cholesterol absorption inhibitors, and other lipid-lowering drugs.

[0122] The optimal therapeutically effective amount of the compounds or compositions of the present disclosure can be determined experimentally, taking into account the exact mode of administration, the form of administration of the drug, the indication for which administration is intended, the subjects involved (e.g., weight, health, age, sex, etc.), and the preference and experience of the attending physician or veterinarian.

[0123] Effective doses and toxicity of the disclosed compounds and compositions that perform well in in vitro testing and have been found to be therapeutically effective in these animal models can be determined in studies using small animal models (e.g., mice, rats) following methods established in the art, and these drugs can be administered by the same routes as proposed for human trials.

[0124] For any pharmaceutical composition used in the methods of the present disclosure, dose-response curves derived from animal systems can be used to determine test doses for human administration. In determining the safety of each composition, the dose and dosing frequency should meet or exceed the dose and frequency expected in any clinical trial.

[0125] As disclosed herein, the dosage of the compounds or compositions of the present disclosure is determined to ensure that the dosage administered continuously or intermittently does not exceed the amount determined after considering the results in the experimental animals and the individual conditions of the patient. The specific dosage will naturally vary (and ultimately be determined according to the judgment of the practitioner and the circumstances of each patient), depending on the dosing procedure, the condition of the patient or subject animal, such as age, weight, sex, sensitivity, dosing, dosage cycle, drugs used in combination, severity of the disease, etc.

[0126] Toxicity and therapeutic efficacy of the compositions of the present disclosure can be determined by standard pharmaceutical procedures in experimental animals, for example, by determining the LD 50 (the dose lethal to 50% of the population) and ED 50 (The dose that is therapeutically effective in 50% of the population). The dose ratio between the therapeutic effect and the toxic effect is the therapeutic index, which can be expressed as ED 50 / LD 50 ratio.

[0127] The compounds of the present disclosure can be formulated for parenteral, oral, topical, transdermal, transmucosal, intranasal, buccal administration, or by any other standard route of administration. Parenteral administration includes intravenous (iv), subcutaneous (sc), intraperitoneal (ip), intramuscular (im), subdermal (sd), intradermal (id), intraarticular, intrasynovial, intraarteriole, intraventricular, intrathecal, intrasternal, intrahepatic, intralesional or intracranial administration, by direct injection, such as by bolus injection, continuous infusion or gene gun, etc. The preferred route of administration according to the present disclosure will depend primarily on the indication being treated, and includes topical, oral, subcutaneous, intravenous and intranasal administration, etc.

[0128] Injectable preparations can be presented in unit dose form, such as in ampoules or multi-dose containers, and preservatives are added. The composition can be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain a formulation agent, such as a suspending agent, a stabilizer, and / or a dispersant. Alternatively, the active ingredient can be present in powder form and reconstituted with a suitable vehicle, such as sterile pyrogen-free water, before use. Preparations suitable for parenteral administration may contain substances that increase viscosity, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. Alternatively, the preparation may also contain a stabilizer. In addition, the compounds of the present disclosure may also be encapsulated in liposomes for administration. The compound may be present in both the aqueous layer and the lipid layer, or in a so-called liposomal suspension, depending on its solubility. The hydrophobic layer generally includes, but is not limited to, phospholipids (such as lecithin and sphingomyelin), steroids (such as cholesterol), more or less ionic surfactants (such as diacetyl phosphate, stearylamine, or phosphatidic acid), and / or other hydrophobic materials.

[0129] In a specific embodiment, the compounds and / or compositions of the present disclosure are formulated for oral administration. For oral administration, the preparations of the present disclosure can be in the form of, for example, tablets or capsules, which are prepared by conventional means using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods known in the art. The compositions of the present disclosure can also be introduced into microspheres or microcapsules, for example, made of polyglycolic acid / lactic acid (PGLA) (see, U.S. Patent Nos. 5,814,344; 5,100,669 and 4,849,222; PCT Publication Nos. WO 95 / 11010 and WO 93 / 07861). Liquid preparations for oral administration can be in the form of, for example, solutions, syrups, emulsions or suspensions, or they can be presented as dry products, reconstituted with water or other suitable vehicles before use. Such liquid preparations can be prepared by conventional means together with pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, ethanol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl parabens or sorbic acid). Preparations can also contain buffer salts, flavoring agents, coloring agents and sweeteners where appropriate. Orally administered preparations can be appropriately formulated to obtain controlled release of active compounds.

[0130] For administration by inhalation, therapeutics according to the present disclosure may conveniently be delivered in the form of an aerosol spray from a pressurized pack or nebulizer, using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may 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 may be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0131] In addition to the aforementioned preparations, the composition can also be formulated as a storage preparation. Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the compound can be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, such as a sparingly soluble salt. Example

[0132] The following examples are provided to further describe some embodiments disclosed herein. These examples are intended to illustrate, but not to limit, the disclosed embodiments. Example 1. Endothelial GR deficiency leads to a fibrogenic phenotype in the kidneys of diabetic mice

[0133] Streptozotocin (STZ)-induced diabetic CD-1 mice are an established mouse model for studying diabetic nephropathy. 29-31 , because the renal fibrosis phenotype is mouse strain specific 30 Although STZ-induced diabetic CD-1 mice and diabetic C57BL / 6 mice exhibit similar blood glucose levels, kidneys from diabetic CD-1 mice have been shown to have a higher incidence of EndMT and more severe fibrosis compared with kidneys from diabetic C57BL / 6 mice. 29,32 Therefore, the diabetic CD-1 mouse is considered a profibrotic strain, while the diabetic C57B / L6 mouse is considered a less fibrotic strain. 32,33 .

[0134] Kidneys from diabetic CD-1 mice showed a significant suppression of GR compared with kidneys from diabetic C57BL / 6 mice as assessed by immunofluorescence staining ( Figure 1 A). Furthermore, ECs isolated from kidneys of diabetic CD-1 mice showed a significant suppression of GR expression at both protein and mRNA levels compared with diabetic C57BL / 6 mice and non-diabetic controls of both genotypes ( Figure 1 B). The effect of corticosterone deficiency on the progression of renal fibrosis was evaluated by bilateral adrenalectomy in both strains of mice. Corticosterone levels were significantly suppressed in all mice studied, confirming the efficacy of the surgical procedure ( Fig. 9 A); adrenalectomy has no effect on blood sugar ( Fig. 9 B). Adrenalectomy did not result in any significant differences in the degree of fibrosis or collagen expression levels ( Fig. 9 C), indicating that global inhibition of corticosterone had no effect on the observed fibrotic phenotype. Example 2. Loss of EC GR exacerbates renal fibrosis

[0135] To verify that GR knockout (GR ECKO ) mice, the inventors performed Western blotting and qPCR on the GR in isolated renal endothelial cells. Flow cytometry analysis showed that renal EC isolation was completed with a purity of approximately 95% ( Fig.10 ).like Fig.11 A- 11B, as expected, in the ECKOIn the EC of 8-week-old GR mice, the mRNA and protein levels were significantly decreased. ECKO (GR fl / fl ; Tie1 Cre+) and Cre- littermate controls (GR fl / fl ) and GR fl / fl ; Tie1 Cre+ / Apoe - / - (DKO) mice and Cre- littermates (GR fl / fl ;Apoe - / - ) were injected with low-dose STZ (50 mg / kg / day IP) for 5 times to produce diabetes ( Figure 2 A). Animals were monitored for 4 months after STZ treatment before being euthanized. At the time of euthanasia, non-diabetic and diabetic GR ECKO There were no significant changes in body weight, blood glucose, heart weight, liver weight, triglycerides, or cholesterol in diabetic GR and DKO mice and their littermate controls; however, EC KO Kidney weights and albumin / creatinine ratios were relatively higher in DKO and diabetic DKO compared with their respective diabetic controls. Figure 2 B-2I, Fig.12 Systolic blood pressure in diabetic mice of all genotypes was approximately 20 mm Hg lower than that in corresponding nondiabetic controls, probably due to the severe diuresis induced by diabetes, but otherwise there were no significant differences ( Fig.12 ). and diabetes GR EC KO Kidney weight and albumin / creatinine ratio were significantly increased in diabetic DKO compared with those in diabetic littermates. Renal fibrosis was assessed by histological analysis of kidney sections from all genotypes. ECKO Mice showed higher relative fibrosis area, higher relative collagen deposition, and more severe glomerulosclerosis at the 4-month time point ( Figure 2 J. Fig.13 A) and diabetes Apoe - / - Control and diabetic GR ECKO In contrast, diabetic DKO showed a substantial increase in the relative area of ​​fibrosis and relative collagen deposition ( Figure 2 J) Compared with diabetic littermates, diabetic GR ECKO Mice showed increased expression of vimentin, snail1, and HIF1α, and decreased expression of PPARα at the 4-month time point ( Fig.13 B; Fig.14 ). and diabetes Apoe - / - A similar pattern was observed in diabetic DKO compared with controls ( Fig.13 B; Fig.14 ). Immunofluorescence data showed that in the presence of GRECKO The deposition of type I collagen and fibronectin was higher in the kidneys of diabetic animals with the highest deposition observed in DKO mice ( Fig.15 ).

[0136] To test the role of endothelial GR in nondiabetic fibrosis, 8-week-old GR ECKO and control littermates underwent unilateral ureteral obstruction (UUO) ( Fig.16 A) Control and GR ECKO There were no significant differences in renal fibrosis between the contralateral kidneys of mice. However, compared with the UUO kidneys of littermate control mice, GR ECKO The UUO kidneys of mice showed a larger relative area of ​​fibrosis and more collagen deposition ( Fig.16 B) Immunofluorescence staining showed that GR ECKO There was more deposition of type I collagen, αSMA, and fibronectin in the UUO kidneys ( Fig.16 C). In C57BL / 6 mice subjected to UUO, administration of the GR agonist dexamethasone abolished the renal fibrogenic phenotype by day 10 ( Fig.17 A). Dexamethasone treatment slightly suppressed renal fibrosis in the contralateral kidney of C57BL / 6 mice at day 15, but did not induce any significant changes in non-diabetic control CD-1 mice ( Fig.17 A-17B). Example 3. Endothelial GR deficiency reprograms cytokine and chemokine homeostasis

[0137] Inflammation is a key factor in fibroblast activation in the kidneys of diabetic mice 34,35 , and disruption of cytokine and chemokine homeostasis contributes to the development of diabetic nephropathy 36-38 To investigate whether homeostasis is dysregulated in this model, cytokine analysis was performed on plasma from diabetic mice with severe fibrosis (diabetic CD-1) and plasma from diabetic mice with a less severe degree of fibrosis (diabetic C57BL / 6). Compared with plasma from diabetic C57BL / 6 mice, diabetic CD-1 mice exhibited higher levels of plasma IL-1β, IL-6, IL-10, IL-17, G-CSF, IFN-γ, TNF-α, MCP-1, CCL3, and CCL4, but suppressed levels of CCL5 ( Fig.18 ). In the GR from diabetic and non-diabetic ECKO mice and littermate control mice as well as diabetic and nondiabetic DKO and Apoe - / -The same cytokines were also analyzed in the plasma of control mice. A similar pattern was observed in both genotypes, as there was a significant increase in the expression of cytokines in diabetic GR compared to the plasma of their respective diabetic control littermates. ECKO In the plasma of diabetic and DKO mice, IL-1β, IL-6, IL-10, eotaxin, G-CSF, and CCL4 were significantly increased, while CCL5 was significantly decreased ( Figure 3 A; Fig.19 A). Similarly, mRNA gene expression analysis showed that GR ECKO In the kidneys of diabetic DKO and diabetic mice, the levels of IL-1β, IL-6, IL-10, IL-17, eotaxin, and CCL4 were significantly upregulated, whereas the level of CCL5 was significantly downregulated ( Fig.19 B), showing that EC inflammation is greater in mice lacking endothelial GR.

[0138] Stimulation of renal endothelial cells isolated from CD-1 mice with recombinant IL-1β, IL-6, TNFα, and TGFβ resulted in a significant increase in the expression of the mesenchymal markers vimentin and αSMA ( Fig. 20 A-20B); Neutralization of IL-6 using IL-6 neutralizing antibodies inhibited the expression of vimentin in TGFβ2-treated and untreated cells ( Fig. 20 C). To further investigate the role of IL-6 in diabetes-associated renal fibrosis, the present inventors injected (IP) IL-6 neutralizing antibodies in non-diabetic and diabetic CD-1 mice. In non-diabetic mice, IL-6 neutralization did not cause any significant difference; however, in diabetic mice, IL-6 neutralization resulted in a significant reduction in fibrosis and collagen deposition ( Fig. 20 D) In ​​addition, from the diabetes GR ECKO Cells isolated from DKO and DKO mice expressed higher levels of IL-6 in the culture medium compared with cells isolated from their respective diabetic control littermates ( Figure 3 B) When the non-diabetic and diabetic GR ECKO When IL-6 neutralization was performed in DKO and DKO mice, it completely reversed the fibrogenic phenotype in diabetic animals compared with mice injected with the corresponding IgG control ( Figure 3 C). No significant effects were observed in non-diabetic mice ( Fig.21 ). Example 4. Canonical Wnt signaling is a new drug target for endothelial GR action

[0139] Given the recently described regulation of Wnt signaling by endothelial GR 27 and the well-established role of Wnt signaling in renal fibrosis39 The present inventors evaluated the relationship between the GR ECKO mRNA expression of Wnt-dependent genes and fibrogenic markers in ECs isolated from the kidneys of diabetic DKO mice and their diabetic littermates. ECKO In the kidneys of diabetic DKO mice, the expression of Wnt-dependent genes and fibrogenic markers was upregulated compared with their respective controls. However, the kidneys of diabetic DKO mice showed the highest expression of Wnt-dependent genes (such as axin2 and tcf) and fibrogenic markers (such as αSMA and fibronectin) ( Fig.14 A) In diabetic GR ECKO In ECs isolated from kidneys of diabetic and DKO mice, the expression of HIF1α and Snail1 was upregulated, while the expression of PPARα was downregulated compared with that of diabetic controls ( Fig. 22 A) In ECs isolated from cultured glomeruli, diabetic GR cells were expressed in comparison with littermate control and nondiabetic mice. ECKO The expression of Wnt-dependent genes Axin2 and TCF as well as fibronectin is upregulated in glomerular ECs of mouse kidneys ( Fig. 22 B) In diabetic GR ECKO The protein expression of β-catenin was also elevated in the renal interstitial and glomerular compartments of diabetic and DKO mice compared with their respective controls ( Fig. 22 C) Western blot showed that in diabetic GR ECKO In ECs isolated from diabetic mice, protein expression of vimentin, HIF1α, snail1, and active β-catenin was increased, whereas levels of PPARα and CPT1a were decreased compared with those of diabetic control littermates ( Figure 4 A). Compared with littermates of Apoe- / - and diabetic GR ECKO ECs isolated from diabetic DKO mice showed higher levels of fibronectin, αSMA, and active β-catenin compared to those from diabetic DKO mice ( Figure 4 B).

[0140] The same pattern was observed using immunofluorescence co-staining, where diabetic GR ECKO and diabetic DKO mice showed higher levels of αSMA / CD31 and TGFβR1 / CD31 co-staining in the kidney compared with their respective controls, suggesting the occurrence of EndMT ( Figure 4 C) Compared with control EC, diabetic GR ECKO There was no difference in endothelial cell density between diabetic and DKO kidney ECs; however, as shown by FITC-dextran staining, endothelial cell density was significantly higher in diabetic GR ECKOIn kidneys and ECs isolated and cultured from DKO and diabetic mice, endothelial cell permeability was higher than that of their respective control littermates ( Fig.23 A-23C). Example 5. Inhibition of canonical Wnt signaling improves renal fibrosis

[0141] Kidneys from diabetic CD-1 mice showed a significant increase in the expression of β-catenin, a marker of canonical Wnt signaling, compared with kidneys from diabetic C57BL / 6 mice, as assessed by immunohistochemical staining ( Fig.24 A). Kidneys from diabetic CD-1 mice also showed a substantial increase in mRNA expression of Wnt-dependent genes and fibronectin compared with kidneys from diabetic C57BL / 6 mice and nondiabetic control mice of both genotypes ( Fig.24 B) To determine whether inhibition of the Wnt signaling pathway could alleviate the observed fibrosis, the inventors utilized LGK974, a small molecule inhibitor of all secreted Wnts. 40 . Fig.24 CD depicts a schematic diagram showing the experimental protocol for LGK974 treatment in diabetic CD-1 and UUO mice. In both models used, LGK974 significantly reduced ECM deposition, relative area of ​​fibrosis, collagen accumulation, and glomerular sclerosis ( Fig.24 E-24F). Wnt inhibition also substantially restored endothelial GR and suppressed β-catenin levels in diabetic and UUO mice ( Fig.24 In diabetic CD-1 mice, LGK974 also significantly suppressed the increase in IL-1β, IL-6, IL-10, G-CSF, TNFα, MCP-1, and CCL4 levels, while increasing the level of CCL5 ( Fig.25 ). Example 6. Wnt inhibitors partially inhibit diabetic GR ECKO Fibrogenic phenotype in the kidney

[0142] To determine whether Wnt inhibition could alleviate the GR ECKO To investigate the renal fibrosis observed in mice, one group of animals was treated with LGK974. At 8 weeks of age, control and GR ECKO Mice were injected with STZ 50 mg / kg for 5 consecutive days. At 16 weeks after injection, LGK974 was administered by oral gavage for another 8 weeks ( Figure 5 A). At the time of euthanasia, there were no significant differences in body weight or blood glucose between the groups ( Figure 5 B; Fig.26 ). However, in diabetic GR treated with Wnt inhibitors ECKO A significant reduction in kidney weight was observed in diabetic mice and control mice ( Figure 5B) Wnt inhibition significantly improved the relative area of ​​fibrosis, relative collagen deposition, and tubular damage in diabetic control mice; this effect was less prominent but was observed in diabetic GR mice. ECKO Still significant in mice ( Figure 5 C) In non-diabetic control mice or non-diabetic GR ECKO In diabetic mice, Wnt inhibition did not induce any prominent changes in the renal fibrogenic phenotype or β-catenin levels compared with their untreated nondiabetic controls ( Fig. 27 ). Wnt inhibition suppresses diabetic GR ECKO The expression of cochlear protein 1 and HIF1α in the kidney was significantly increased, and the level of PPARα was significantly increased ( Fig.28 Similar effects were observed in isolated EC ( Fig.29 Wnt inhibition also significantly suppressed EndMT (CD31 / αSMA double-positive cells) in diabetic control mice; this effect was not observed in diabetic GR ECKO Less prominent in mice ( Fig.30 ). However, Wnt inhibition significantly reduced both control and diabetic GR ECKO EMT (E-cadherin / αSMA double positive cells) levels in mice ( Fig.30 ). Example 7. Metabolic reprogramming caused by endothelial GR deficiency accelerates renal fibrosis

[0143] There is increasing recognition that central metabolic defects promote renal fibrosis 32,41 Defective fatty acid (FA) metabolism in ECs leads to EndMT events 42 To investigate whether FA metabolism is disturbed in this model, radiolabeled [ 14 C] Palmitate uptake experiment. The present inventors observed that FA uptake was higher in EC isolated from diabetic kidneys of a strain with a higher degree of fibrosis (diabetic CD-1) compared to EC from kidneys of a strain with a lower degree of fibrosis (diabetic C57BL / 6). Administration of Wnt inhibitors inhibited FA uptake in EC of CD-1 mice ( Fig.31 A) Renal ECs from diabetic GR compared with those from diabetic control littermates. ECKO and DKO mice showed higher FA uptake in renal ECs ( Figure 6 A). We also measured radiolabeled [ 14 C] palmitate 14 FA oxidation (FAO) was assessed by CO2 release. FAO was reduced in renal ECs isolated from diabetic CD-1 mice, whereas Wnt inhibition restored FAO levels ( Fig.31B) From diabetes GR ECKO Cultured renal ECs from diabetic DKO mice showed reduced FAO levels compared with their diabetic control littermates ( Figure 6 B) In addition, from the control and GR ECKO Gene expression of fatty acid transporters 1 and 4 (FATP1 and FATP4) was unchanged in both nondiabetic and diabetic ECs isolated from mouse kidneys; however, gene expression levels of another fatty acid transporter, CD36, were reduced in diabetic mice of both genotypes compared with nondiabetic controls ( Fig.32 ).

[0144] In a subsequent set of experiments, diabetic CD-1 mice were treated with the FA synthase inhibitor C75, the FAO inhibitor etomoxir, the PPARα agonist fenofibrate, or the cholesterol-lowering drug simvastatin for 4 weeks. Fenofibrate and C75 alleviated the fibrogenic phenotype, while etomoxir exacerbated fibrosis. Simvastatin treatment did not cause a significant suppression of fibrosis levels. Fig.33 A). Fenofibrate and C75 restored GR protein levels in CD31-positive cells, whereas etomoxir suppressed them, and simvastatin did not show any substantial effect ( Fig.33 B). In diabetic kidneys, fenofibrate and C75 downregulated fibronectin and αSMA mRNA levels, whereas etomoxir upregulated this mRNA, and simvastatin did not cause any significant changes in fibronectin or αSMA gene expression levels ( Fig.33 C). These FA modulators did not induce any significant differences in blood glucose levels ( Fig.33 D) Etomoxir treatment induces FAO in diabetic CD-1 mice (by [ 14 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 ( Fig.33 E-33F).

[0145] In non-diabetic and diabetic control littermates as well as GR ECKO Etomoxir and C75 ( Figure 6 C-6D; Fig.34 ; Fig.35 ). After treatment with etomoxir or C75, non-diabetic or diabetic control littermates and GR ECKO There were no significant differences in body weight, blood glucose, or kidney weights between the mice ( Figure 6 C; Fig.34Data from kidney EC showed that in non-diabetic control littermates, etomoxir caused a significant suppression of FAO, whereas C75 restored FAO ( Fig.34 ). However, in the diabetic GR ECKO In mouse kidney ECs, etomoxir caused a significant inhibition of FAO, which was not rescued by C75 ( Figure 6 c) In diabetic controls and diabetic GR ECKO In the kidneys of diabetic control mice, etomoxir treatment accelerated the renal fibrogenic phenotype, suppressed CPT1a levels, and increased β-catenin expression levels. In the kidneys of diabetic control mice, C75 treatment significantly abolished the renal fibrogenic phenotype, restored CPT1a, and completely reduced β-catenin levels. ECKO These effects were also observed in mice, but to a lesser extent. ( Figure 6 D) Etomoxir slightly increased the expression of GR in non-diabetic controls and ECKO renal fibrosis in mice, which was alleviated by C75 ( Fig.35 ). Etomoxir reduced CPT1a expression in diabetic control mice and also increased β-catenin expression; C75 was able to substantially reverse both effects. ECKO A similar pattern was observed in mice, but also to a lesser extent ( Figure 6 E). Example 8. GR deficiency-associated EndMT disrupts central metabolism and induces mesenchymal transition in renal tubular epithelial cells change

[0146] These in vivo data suggest that GR ECKO Mice showed enhanced EndMT and EMT in their diabetic kidneys. GR overexpression suppressed the elevated expression of αSMA, type I collagen, and β-catenin in HUVECs stimulated by TGFβ2 ( Fig.36 To test whether endothelial GR deficiency affects the mesenchymal program and leads to defects in central metabolism in adjacent epithelial cells, the inventors analyzed the effect of conditioned medium from GR knockdown HUVEC on the mesenchymal phenotype of HK-2 cells ( Figure 7 A). Conditioned medium (CM) from GR siRNA-transfected HUVECs decreased the level of E-cadherin and increased the protein levels of αSMA, TGFβR1, and β-catenin in HK-2 cells compared with the medium from scrambled siRNA-transfected HUVECs ( Figure 7 B-7C). Treatment with CM from GR siRNA-transfected HUVECs resulted in a significant decrease in FAO levels, oxygen consumption rate, and cellular ATP levels in HK-2 cells ( Figure 7D-7F). TECs incubated with CM from HUVECs treated with control siRNA showed significant downregulation of mRNA expression of FAO-responsive genes Cpt1a, Cpt2, Pparα, and Pgc1α compared with TECs incubated with CM from HUVECs treated with control siRNA ( Figure 7 G).

[0147] To confirm these in vitro results, diabetic control and diabetic GR ECKO Primary ECs were isolated from mice to analyze the effects of GR-deficient ECs on mesenchymal activation of renal tubular epithelial cells (TECs). ECKO CM from cultured ECs isolated from kidneys of diabetic and control littermates was transferred to cultured TECs from diabetic and control mice ( Figure 7 H). This treatment of CM from GR-deficient cells resulted in a significant suppression of E-cadherin, CPT1a, and PPARα in TECs, and induced the protein levels of αSMA, TGFβR1, and active β-catenin ( Figure 7 I). In addition, from diabetes GR ECKO CM of mouse EC significantly downregulated the mRNA expression of FAO-responsive genes Cpt1a, Cpt2, and Pparα ( Figure 7 J).

[0148] The examples provided above demonstrate that endothelial GR plays a crucial role in regulating fibrogenic processes in a mouse model of diabetic nephropathy. The results show that endothelial GR regulates the mesenchymal transdifferentiation process by affecting FA metabolism and control of canonical Wnt signaling in the kidneys of diabetic mice. It is clear from these data that GR deficiency is one of the catalysts for renal fibrosis in diabetes, which leads to disruption of cytokine and chemokine homeostasis by upregulating canonical Wnt signaling. Ultimately, these processes shift the metabolic switch in favor of FA metabolic defects and associated mesenchymal activation in TECs.

[0149] Metabolic reprogramming in ECs is a key event during myofibroblast formation, proliferation, and development of fibrosis in diabetic kidneys 12 ,17,41,43 ,44 . Data suggest that GR deficiency is a critical step in metabolic reprogramming of renal ECs. Although bilateral adrenalectomy significantly suppressed corticosterone in all mice studied, this global absence of systemic steroid signaling was insufficient to alter fibrotic progression in diabetic mice, suggesting that tissue-specific effects of targeted GR deficiency in endothelial cells supersede systemic effects; this phenomenon requires further investigation. GR ECKOChanges in cytokine levels in mouse plasma included increased levels of proinflammatory cytokines (IL-1β, IL-6, and IL-17) and the anti-inflammatory cytokine IL-10. To date, the role of IL-10 in renal fibrosis in diabetic nephropathy has not been fully investigated. However, these data suggest that IL-6 is a key inflammatory cytokine that is elevated in the context of endothelial GR inhibition. Neutralization of IL-6 in diabetic mice completely reversed the renal fibrotic phenotype, suggesting that IL-6 has a key profibrotic role in diabetes.

[0150] Recently, it has been shown that loss of endothelial GR leads to upregulation of canonical Wnt signaling 27 It is generally believed that GR exerts its anti-inflammatory effect by targeting the NF-kB signaling pathway. 45 However, in ECs GR also targets canonical Wnt signaling, independent of its traditional target NF-kB 27,45 Inhibition of Wnt signaling in ECs may prove to be a valuable therapeutic opportunity to combat diabetic nephropathy. The Wnt pathway is known to be an important contributor to renal fibrosis, and activation of canonical Wnt signaling results in disruption of cytokine and chemokine homeostasis. 39,46-48 These data suggest that higher levels of GR-deficient canonical Wnt signaling are associated with the induction of mesenchymal and fibrogenic markers.

[0151] To further test the therapeutic potential of Wnt inhibition, the inventors used the small molecule LGK974. Wnt inhibition clearly inhibited canonical Wnt signaling, substantially improved the fibrogenic phenotype in a mouse model of diabetic nephropathy, and restored endothelial GR levels. These data suggest that tonic inhibition of canonical Wnt signaling in ECs is one mechanism by which GR exerts its anti-fibrotic effects. Notably, this effect was not observed in GR ECKO This was less evident in mice, probably because Wnt inhibition was able to suppress EMT in other cell types (TECs), but it was unable to alleviate EndMT. Collectively, these data suggest that endothelial GR is a key anti-EndMT molecule.

[0152] Few studies have explored lipid metabolism in kidney cells 49,50 Central metabolic defects promote diabetic nephropathy 32,41 , and some reports suggest that changes in cytokine levels can affect renal lipid metabolism in diabetic nephropathy 51,52 Clinical observations suggest a potential link between lipid levels and kidney disease 53 , and lipid control appears to be important in the prevention and treatment of diabetic nephropathy 49,50Here, the present inventors aimed to dissect the role of lipid metabolism in ECs in the regulation of diabetic nephropathy.

[0153] Apoe - / - Compared with WT mice, DKO mice showed exacerbated atherosclerosis, which could not be explained by differences in plasma lipid levels. 25 This observation prompted the evaluation of the diabetic phenotype in these animals. It is clear from the data that hypercholesterolemia exacerbates GR ECKO The severity of renal fibrosis in the diabetic nephropathy mouse model was significantly increased, suggesting that hypercholesterolemia affects EC metabolism and promotes renal fibrosis. However, similar to existing clinical data, the cholesterol-lowering drug simvastatin did not alleviate the severity of renal fibrosis in this diabetic nephropathy mouse model. Interestingly, fibrates are a class of drugs that treat hypertriglyceridemia with elevated non-HDL cholesterol remnants. However, the role of fibrates in patients with diabetic nephropathy has not been established. 50,63 .

[0154] The present inventors evaluated the role of Wnt activation associated with endothelial GR deficiency and its relevance to defective FA metabolism. Disruption of endothelial FA metabolism contributes to EndMT activation in diabetic kidneys 8,31,64 . FAO activation produced a significant inhibitory effect on fibrosis by restoring endothelial GR levels in diabetic mice. Conversely, FAO inhibition led to accelerated fibrosis by reducing endothelial GR levels in diabetic control mice, indicating that endothelial GR is a key protein for the action of FAO modulators. These data clearly demonstrate that the antifibrotic effects of the FAO activator C75 are dependent on endothelial GR, which in turn indicates that EC is required for the antifibrotic effects of this drug.

[0155] When the GR from diabetes ECKO When the conditioned medium of GR-deficient EC of mice was transferred to cultured TEC from diabetic control kidneys, the inventors observed the induction of mesenchymal markers, the activation of TGFβ and canonical Wnt signaling, and the inhibition of epithelial cell markers. These findings suggest that EndMT leads to the activation of the EMT process in diabetes. GR-deficient cells have higher levels of TGFβ-smad3 and canonical Wnt signaling, which are associated with perturbations in plasma cytokine levels and inhibition of FAO. The cumulative effect of these metabolic changes leads to the activation of mesenchymal transformation in EC, which appears to have a paracrine effect on neighboring TEC. The functional importance of GR protein in EC homeostasis is graphically depicted ( Figure 8 ).

[0156] GR agonists such as dexamethasone activate GR signaling in all cell types; however, in diabetes, dexamethasone intervention is not preferred because it causes severe and predictable exacerbations of hyperglycemia. These data highlight a regulatory role for GR in ECs.

[0157] Together, these findings highlight the regulatory role of GR on EndMT in the diabetic kidney, which is mediated through control of canonical Wnt signaling and associated defective FA metabolism. This study provides insights into the biology of GR and its key role in renal fibrosis and diabetes.

[0158] The following is the method used in the above examples.

[0159] Reagents and antibodies. Rabbit polyclonal anti-GR (SAB4501309), mouse monoclonal anti-αSMA (Cat. No. A5228), and mouse monoclonal anti-β-actin (AC-74) (A2228) antibodies were from Sigma (St Louis, MO). Anti-TGFβR1 (ab31013), PPARα (ab215270), mouse monoclonal anti-vimentin (RV202) (ab8978), rabbit polyclonal anti-αSMA (ACTA2) (ab5694), anti-HIF1α (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-phosphorylated (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).

[0160] Animal experiments. All experiments were performed according to protocols approved by the Institutional Animal Use and Care Committee of the Yale University School of Medicine and in accordance with the guidelines for laboratory animal care of the National Institutes of Health (NIH). Mice were housed at an ambient temperature of 68-79°F with a humidity range of 30%-70%. They were exposed to a 12-hour light-dark cycle. ECKO ) and mice lacking this receptor in an Apoe-deficient background (DKO) 25 ; The mice were of C57BL / 6 background. Diabetes was induced in 10-week-old male mice by five consecutive intraperitoneal (IP) administrations of streptozotocin (STZ) 50 mg / kg in 10 mmol / L citrate buffer (pH 4.5). Wnt inhibitor (LGK974) was given to diabetic mice treated with STZ for 16 weeks at a dose of 5 mg / kg, with a frequency of 6 doses per week for 8 weeks. 40 . Give GR ECKO and control littermates were dosed (IP) three times per week with etomoxir (20 mg / kg) and C75 (15 mg / kg) for 3 weeks.

[0161] A single IP dose of 200 mg / kg STZ was used to induce diabetes in CD-1 mice.

[0162] In one experiment, male mice were randomly assigned to one of the following 4 groups at 16 weeks after induction of diabetes: (i) untreated, (ii) fenofibrate (100 mg / kg), (iii) simvastatin (40 mg / kg), or (iv) a 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 the following 3 groups: (i) untreated (vehicle), (ii) etomoxir (20 mg / kg), or (iii) C75 (15 mg / kg); in each case, mice were treated three times per week (IP) for a total of 4 weeks.

[0163] In non-diabetic and diabetic mice, IL-6 IgG and control IgG were injected IP three times a week at a dose of 3 mg / kg for a total of four weeks. During the experiment, all mice had free access to food and water. During the experiment, blood was obtained by retro-orbital bleeding. Blood glucose was measured by glucose test strips. Urine albumin levels were determined using a mouse albumin ELISA kit (Exocell, Philadelphia, PA).

[0164] Tissue and blood were collected at the time of euthanasia. 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 cryosectioning or in 4% paraformaldehyde for histological staining.

[0165] Bilateral adrenalectomy. Diabetic mice (24 weeks old) injected with STZ for 16 weeks and non-diabetic CD-1 and C57BL / 6 mice were used for adrenalectomy and sham surgery. The animals were manipulated every day for the week before the experiment to reduce stress reactions. Buprenorphine was used as an analgesic. The first dose was administered 30 minutes before surgery and then every 12 hours for 72 hours at a dose of 0.05 mg / kg subcutaneously. For the first 24 hours after surgery, mice were given drinking water containing 0.9% saline to counteract the effects of mineralocorticoid removal. Adrenalectomized animals were euthanized 4 weeks after surgery. Blood was collected between 9:00 and 10:00 am. Corticosterone levels were measured using an ELISA kit from Cayman Chemical (Ann Arbor MI).

[0166] Mouse model of unilateral ureteral obstruction (UUO). UUO surgery was performed as previously described. 19 . Briefly, mice were anesthetized with isoflurane (3%–5% for induction and 1%–3% for maintenance). The left side of the mouse abdomen was shaved, a vertical incision was made through the skin with a scalpel, and the skin was turned open. A second incision was made through the peritoneum to expose the kidney. The left ureter was ligated twice with surgical silk suture 15 mm below the renal pelvis, and 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 replace fluid loss. The incisions were sutured and the mice were housed in single cages. Buprenorphine was used as an analgesic. The first dose was administered 30 minutes before surgery and then every 12 hours for 72 hours at a dose of 0.05 mg / kg subcutaneously. At 10 days after UUO, the mice were euthanized and kidney and blood samples were harvested after perfusion with PBS. The contralateral kidney was used as a non-fibrotic control in all experiments performed using this model.

[0167] Blood pressure measurement. Measurements were performed using the tail cuff method according to the manufacturer's instructions. Briefly, mice were trained for 5 days before blood pressure measurement. After placing the mice in a restraint platform maintained at 33–34°C, the tails of the mice were passed through an optical sensor and the tail cuff was pressed. The instrument automatically measured blood pressure and repeated 10 times. The data are expressed as the average of 10 measurement cycles.

[0168] Lipid analysis. Mice were fasted for 12-15 hours and blood was collected by retro-orbital venipuncture. Whole blood was centrifuged and plasma was stored at -80°C. Total cholesterol and triglyceride levels were measured enzymatically using kits from Wako and Sigma, respectively, according to the manufacturer's instructions.

[0169] Morphological assessment. The relative area of ​​mesangial matrix was assessed using the point counting method. The inventors analyzed PAS-stained glomeruli from each mouse using a digital microscope screen grid containing 540 (27×20) points. Masson trichrome staining images were evaluated by ImageJ software, and the fibrosis area was estimated.

[0170] Sirius red staining. Dewaxed sections were incubated with Sirius red solution at room temperature for 1 hour. The slides were washed twice with acetic acid solution, each wash for 30 seconds. The slides were then dehydrated three times in anhydrous ethanol, transparentized with xylene, and mounted with synthetic resin. Sirius red staining was analyzed using ImageJ software, and the fibrosis area was quantified.

[0171] Immunohistochemistry. Paraffin-embedded kidney sections (5 μm thick) were dewaxed and rehydrated (2 min in xylene, 4 times; 1 min in 100% ethanol, twice; 1 min in 95% ethanol; 45 s in 70% ethanol; and 1 min in distilled water), and antigens were retrieved in 10 mM citrate buffer, pH 6, at 98°C for 60 min. To block endogenous peroxidases, 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-DO1P; 1:100) antibodies were used. In the negative control, the primary antibody was omitted and replaced with blocking solution.

[0172] Immunofluorescence. Frozen kidney sections (5 μm) were used for immunofluorescence; double positive labeling of 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 air-dried and placed in acetone at -30°C for 10 minutes. Once the sections were dry, they were washed twice in phosphate-buffered saline (PBS) for 5 minutes and then blocked with 2% bovine serum albumin / PBS for 30 minutes at room temperature. Thereafter, the sections were incubated in primary antibodies (1:100) for 1 hour and washed three times in PBS (5 minutes). Next, the sections were incubated with secondary antibodies for 30 minutes, washed three times with PBS (5 minutes each), and mounted with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Immunolabeled sections were analyzed by fluorescence microscopy. For each mouse, images with an original magnification of ×400 were obtained from six different areas and quantified.

[0173] EndMT and EMT detection. Cryosections (5 μm) were used to detect EndMT and EMT. Cells undergoing EndMT were detected by double positive labeling of CD31 (1:100) and αSMA (1:500) and / or TGFβR1 (1:500). Cells undergoing EMT were detected by double positive labeling of E-cadherin (1:500) and αSMA (1:500). Sections were analyzed and quantified by fluorescence microscopy.

[0174] Endothelial cells were separated. According to the manufacturer's instructions, endothelial cells from the kidneys of non-diabetic and diabetic mice were separated using a standardized kit (Miltenyl Biotech, USA). In short, the kidney was separated and chopped into small pieces. By treating the tissue with trypsin and type I collagenase solution, a series of enzymatic reactions were performed to make a single cell suspension. The precipitation was decomposed with CD31 magnetic beads, and cells labeled with CD31 were separated on a magnetic separator. The cells were further purified on a column. The number of cells was counted by a hemocytometer, and the cells were spread on a 0.1% gelatin-coated culture dish. CD31 (BDB553373) (1:100) and CD45 (BDB553079) (1:100) (both from Becton Dickinson (USA)) conjugated with PE were used to measure cell purity by flow cytometry (BD FACSDiva).

[0175] Isolation of renal TECs. After euthanasia, renal TECs were isolated from diabetic GLRs. ECKOThe kidneys of the control littermates were perfused with PBS (10 mL) and then digested with type II collagenase (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 at 37 ° C for another 5 minutes while rotating to release the cells. The digested tissue and cell suspension were passed through a 70-μm cell strainer, centrifuged at 50 g for 5 minutes, and washed 2 times in PBS to collect TEC. The separated TECs were inoculated on collagen-coated culture dishes and cultured in renal epithelial cell culture medium (C-26130, PromoCell) supplemented with growth factors for TEC growth.

[0176] Cellular bioenergetics analysis. FAO-related oxygen consumption rate (OCR) was studied using an extracellular flux assay (Seahorse XFe96, Agilent Technologies). On the day of the assay, the substrate-limited medium was replaced with Krebs-Henseleit buffered assay medium supplemented with 0.2% carnitine and incubated for 1 h at 37°C in the absence of CO2. Finally, BSA or 200 mM palmitate-BSA FAO substrate was added just before starting the assay. After the assay, proteins were extracted from the wells with 0.1% NP-40–PBS solution and quantified using the bicinchoninic acid protein assay (Thermo Fisher Scientific) for data normalization. The OCR was determined by quantifying the Hoechst staining in each well and normalizing the measurements to the cell count. 65 .

[0177] ATP measurement. ATP content was determined using the ATP colorimetric assay kit (Biovision) according to the manufacturer's instructions.

[0178] RNA isolation and qPCR. Total RNA was isolated using a standard Trizol protocol. RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad), and the resulting cDNA was used with qPCR premix and gene-specific primers for qPCR on a Bio-Rad C1000 Touch thermal cycler. Table 1 lists the mouse primers used. 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. Mouse primer sequences

[0179] Western blotting. Protein lysates were boiled in sodium dodecyl sulfate (SDS) sample buffer at 94°C for 5 min. After centrifugation at 17,000 × g for 10 min at 4°C, the supernatant was separated on a 6%-12% SDS polyacrylamide gel and transferred to a PVDF membrane (Immobilon, Bedford, MA) by semi-dry transfer. After blocking with TBS (Tris-buffered saline with 0.05% Tween 20) containing 5% bovine serum albumin (BSA), the membrane was incubated 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 at 4°C overnight. Protein bands were visualized using the Odyssey infrared imaging system (LI-CORBiotechnology) and an enhanced chemiluminescence (ECL) detection system (Pierce Biotechnology, Rockland IL) with ImageQuant LAS 400 (GE Healthcare Life Sciences, Uppsala, Sweden). Densitometry was performed using ImageJ software (NIH).

[0180] In vitro experiments and siRNA transfection. HUVECs were used at passage 4-8 and cultured in endothelial basal medium-2 medium containing growth factors and 10% serum. Human GR-specific siRNA (Invitrogen) at a concentration of 100nM was used for 48 hours to effectively knock down GR. Cells were treated with or without TGFβ2 (10ng / ml) for 48 hours and harvested for western blot analysis. Some transfected cells were treated with fenofibrate (1μM) and etomoxer (40μM) for 48 hours. 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 the cells reached 70% confluence, conditioned medium from control siRNA and GR siRNA-transfected HUVECs was added to HK-2 cell cultures.

[0181] Fatty acid uptake. Cultured isolated renal endothelial cells were incubated with 2 μCi [ 14 C]-palmitate. The [ 14 C]-palmitate uptake.

[0182] Fatty acid oxidation. Cultured isolated renal endothelial cells were incubated with 0.75 mmol / L palmitate (with 2% free fatty acid-free BSA / 2 μCi / mL [ 14 C] palmitate conjugated) medium was incubated for 2 hours. 1 mL of the medium was transferred to a sealable tube with a Whatman filter paper disk in the cap. The trapped molecules in the medium were then released by acidifying the medium with 60% perchloric acid. 14 CO2. The radioactivity adsorbed to the filter disks was then quantified by liquid scintillation counting.

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[0184] The present invention is not limited in scope by the specific embodiments described herein. In fact, in addition to what is described herein, various modifications of the present invention will become apparent to those skilled in the art based on the foregoing description. These modifications are intended to fall within the scope of the appended claims.

[0185] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if actually set forth in this specification.

Claims

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 IL-6 activity inhibitor, wherein the condition or disease is selected from organ fibrosis, kidney disease, dyslipidemia, hypertension, hyperlipidemia, hypercholesterolemia, cardiovascular disease, peripheral arterial 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 renal disease is diabetic nephropathy.

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

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

6. 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 targeting IL-6 receptor.

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 1-3, wherein the inhibitor of IL-6 activity provides a trans-signaling blockade.

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

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

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

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

14. The method of claim 13, wherein the one or more additional agents include one or more of the following: a cholesterol-lowering drug, a blood pressure-lowering therapy, an anti-inflammatory agent, an antithrombotic agent, an anticoagulant, a renin-angiotensin aldosterone system inhibitor (RAAS inhibitor), a beta-adrenergic blocker, a calcium channel blocker, a glucose-lowering drug, and / or a Wnt inhibitor.

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

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

17. The method of claim 14, wherein the hypoglycemic drug 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.

18. The method of claim 14, wherein the Wnt inhibitor comprises a compound having a structure according to Formula (I) or a pharmaceutically acceptable salt thereof: Where X 1 and X 2 Selected from N and CR; X 3 , X 4 , X 5 and X 6 One of them is N, and the others are selected from N and CR; X 7 , X 8 , X 9 and X 10 One of them is N, and the others are selected from N and CR; X 11 , X 12 , X 13 and X 14 One of them is N, and the others are selected from N and CR, and R at each occurrence is independently selected from hydrogen, halogen, cyano, methyl, difluoromethyl and trifluoromethyl. E 19. The method of any one of E 1-18, wherein the subject is a human.

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