TREATMENT OF RENAL DISEASES WITH A BILE ACID DERIVATIVE

BACKGROUND

A gradual decline in renal function occurs even in healthy aging individuals. The fastest growing group of people in the United States with impaired kidney function is the oldest age group. The population older than 65 years in the United States is expected to double in the next 20 years, and that of elderly worldwide is expected to triple from 743 million in 2009 to 2 billion in 2050. This will result in a marked increase in the elderly population with chronic kidney disease (CKD). Moreover, hypertension, obesity, and insulin resistance can induce mitochondrial dysfunction, endoplasmic reticulum stress, oxidative stress, inflammation, altered lipid metabolism, and stimulation of profibrotic growth factors in the kidney, which collectively contribute to age-, diabetes-, and/or obesity-related kidney disease.

The rate of decline in renal function varies by gender, race, and burden of co-morbid conditions. Although greater glomerular, vascular, and interstitial sclerosis is evident on renal tissue examination of healthy kidney donors with increasing age, closer examination of processes leading to sclerosis suggests a role for metabolic and hormonal factors that can decrease the rate of sclerosis. The Baltimore Longitudinal Study of Aging revealed that nearly a third of older healthy adults have little change in renal function over time. Similar findings have also been reported in rodent models of aging.

One of the most successful measures that have been shown to slow down and delay age-related kidney disease is caloric restriction, in part by preventing the increased expression of SREBP-1 and SREBP-2 that are master regulators of fatty acid, triglyceride, and cholesterol synthesis. Prevention of the age-related increases in SREBP-1 and SREBP-2 is associated with decreased renal triglyceride and cholesterol accumulation, decreased renal expression of growth factors, connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF), matrix metalloproteinase inhibitor, and plasminogen activator inhibitor-1 (PAI-1), resulting in prevention of mesangial expansion, podocyte injury and proteinuria. There is a need for therapies for the treatment and prevention of renal diseases, such as age-, diabetes- and/or obesity-related renal diseases. The present application addresses the need. SUMMARY

The present application relates to a method of treating or preventing a renal disease, disorder, or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A:

or a pharmaceutically acceptable salt or amino acid conjugate thereof, wherein:

R1 is C1-C6 alkyl;

R2, R3, and R5 are each independently H or OH; and

R ₄ is CO ₂ H or OSO ₃ H.

The present application also relates to a method of improving kidney function in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof.

The present application also relates to a method of slowing the progress of or reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression in a kidney, or slowing the progress of or reversing age-, diabetes-, and/or obesity- related impairments in mitochondrial biogenesis or mitochondrial function in a kidney, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof. The present application also relates to a method of increasing the level of FXR and/or TGR5 in a kidney in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof.

The present application also relates to a compound of Formula A, or a

pharmaceutically acceptable salt or amino acid conjugate thereof, for treating or preventing a renal disease, disorder, or condition; improving kidney function; slowing the progress of or reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression in a kidney; slowing the progress of or reversing age-, diabetes-, and/or obesity-related impairments in mitochondrial biogenesis or mitochondrial function in a kidney; or increasing the level of FXR and/or TGR5 in a kidney, in a subject in need thereof.

The present application also relates to a compound of Formula A, or a

pharmaceutically acceptable salt or amino acid conjugate thereof, for use in the manufacture of a medicament for the treatment or prevention of a renal disease, disorder, or condition; for improving kidney function; for slowing the progress of or reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression in a kidney; for slowing the progress of or reversing age-, diabetes-, and/or obesity-related impairments in

mitochondrial biogenesis or mitochondrial function in a kidney; or for increasing the level of FXR and/or TGR5 in a kidney, in a subject in need thereof.

The present application also relates to use of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof, in the manufacture of a medicament for the treatment or prevention of a renal disease, disorder, or condition; for improving kidney function; for slowing the progress of or reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression in a kidney; for slowing the progress of or reversing age-, diabetes-, and/or obesity-related impairments in mitochondrial biogenesis or mitochondrial function in a kidney; or for increasing the level of FXR and/or TGR5 in a kidney, in a subject in need thereof.

In one embodiment, a com ound of Formula A is Com ound 1:

or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, a pharmaceutically acceptable salt of Compound 1 is the sodium salt of Compound 1 (i.e., Compound 1-Na). In yet another embodiment, a pharmaceutically acceptable salt of Compound 1 is the triethylammonium salt of Compound 1 (i.e., Compound 1-TEA).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In the case of conflict, the present specification, including definitions, will control. In the specification, the singular forms also include the plural unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. The references cited herein are not admitted to be prior art. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the application will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a bar graph showing relative mRNA level of FXR in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), or 24-month-old mice fed with caloric restriction (24mo-CR). Figure 1B is a bar graph showing relative mRNA level of TGR5 in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 1C is a Western blot showing relative protein level of FXR in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 1D is a bar graph showing relative protein level of FXR in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 2A is a bar graph showing the ratio of albumin/creatinine in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 2B depicts fluorescent images of synaptopodin in young mice fed ad lib (AL- young), old mice fed ad lib (AL-old), old mice fed with caloric restriction (CR-old), or old mice fed ad lib and treated with a compound of the present application (AL-old-INT).

Figure 2C depicts bar graphs showing relative mRNA level of TGF-β (top), fibronectin (middle), or FSP-1 (bottom) in 5-month-old mice fed ad lib (5mo-AL), 24-month- old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 2D depicts immunofluorescent images of fibronectin protein in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month- old mice fed with caloric restriction (24mo-CR).

Figure 3A is a bar graph showing mitochondria to nuclear DNA ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month- old mice fed with caloric restriction (24mo-CR).

Figure 3B is a bar graph showing relative mRNA level of Nrf-1 in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3C is a Western blot showing relative protein levels of pAMPK and AMPK in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month- old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3D is a bar graph showing pAMPK to AMPK ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3E is a bar graph showing relative mRNA level of Sirt1 in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3F is a bar graph showing relative mRNA level of ERRα in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3G is a Western blot showing relative protein levels of PGC1α, Sirt3, and MCAD in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo- AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3H is a bar graph showing Sirt3 to β-actin ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3I is a bar graph showing relative mRNA level of Sirt3 in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR). Figure 3J is a bar graph showing PGC1α to β-actin ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3K is a bar graph showing relative mRNA level of PGC1α in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3L is a bar graph showing MCAD to β-actin ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3M is a Western blot showing the relative protein levels of acetyl-IDH2 and IDH2 in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo- AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3N is a bar graph showing acetyl-IDH2 to IDH2 ratio in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3O is a bar graph showing Complex I activity in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 3P is a bar graph showing Complex IV activity in 5-month-old mice fed ad lib (5mo-AL), 24-month-old mice fed ad lib (24mo-AL), 24-month-old mice fed ad lib and treated with a compound of the present application (24mo-AL-INT), or 24-month-old mice fed with caloric restriction (24mo-CR).

Figure 4 depicts bar graphs showing relative mRNA levels of TNF-α (top), TLR2 (middle), and TLR4 (bottom) in human podocytes treated with serum from 4-month-old mice (Young), 28-month-old mice (Old), or 28-month-old mice treated with a compound of the present application (Old-INT).

Figures 5A-5I are bar graphs showing the relative mRNA levels of FXR (Figure 5A), TGR5 (Figure 5B), Nrf1 (Figure 5C), Sirt1 (Figure 5D), PGC1α (Figure 5E), ERRα (Figure 5F), Sirt3 (Figure 5G), Cox4 (Figure 5H), and LCAD (Figure 5I) in Ames dwarf mice.

Figure 6A is a bar graph showing glomerular and tubular FXR mRNA levels in kidney biopsy samples obtained from human subjects with diabetic- and obesity-related kidney disease, with glomerular and tubular cells obtained by laser capture microdissection.

Figure 6B are immunohistochemical staining images of kidney biopsy samples obtained from healthy human subjects (left) and human subjects with diabetic kidney disease (right).

Figure 6C is a graph displaying FXR protein expression in kidney biopsy samples obtained from human subjects with diabetic kidney disease as determined by

immunohistochemistry.

Figure 6D is a graph displaying TGR5 protein expression in kidney biopsy samples obtained from human subjects with diabetic kidney disease as determined by

immunohistochemistry.

Figure 7A is a Venn diagram displaying transcript numbers regulated by Compound 1- Na compared with Compound 2 or Compound 3 as determined by RNA-Seq analysis.

Figure 7B is a graph displaying activated pathways enriched in DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2 but not enriched in DBA/2J mice with STZ-induced hyperglycemia treated with Compound 3 as determined by RNA-Seq analysis.

Figure 7C is a graph displaying activated pathways enriched in DBA/2J mice with STZ-induced hyperglycemia treated with Compound 3 but not enriched in DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2 as determined by RNA-Seq analysis.

Figure 7D is a graph displaying additional activated pathways enriched in DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na.

Figure 7E is a Western blot showing relative protein level of nSREBP-1 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3. Figure 7F is a bar graph showing relative protein level of the ratio of active SREBP- 1/β-actin in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced

hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 7G is a bar graph showing relative mRNA level of SCD-1 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 7H is a bar graph showing relative mRNA level of SCD-2 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 7I is a bar graph showing relative mRNA level of Fit-1 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 2, Compound 1-Na, or Compound 3. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 7J is a Western blot showing relative protein level of Sirt1 in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3.

Figure 7K is a bar graph showing relative protein level of the ratio of Sirt1/β-actin in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3. ^† P < 0.05 versus db/db, *P < 0.05 versus db/m.

Figure 7L is a Western blot showing relative protein level of PGC-1α in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3.

Figure 7M is a bar graph showing relative protein level of the ratio of PGC-1α/β-actin in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3. ^† P < 0.05 versus db/db, *P < 0.05 versus db/m. Figure 7N is a Western blot showing relative protein level of ERRα in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3.

Figure 7O is a bar graph showing relative protein level of the ratio of ERRα /β-actin in non-diabetic mature mice (db/m), diabetic mice (db/db) and diabetic mice (db/db) treated with Compound 2, Compound 1-Na, or Compound 3. ^† P < 0.05 versus db/db, *P < 0.05 versus db/m.

Figure 7P is a schematic diagram displaying that Compound 1-Na can simultaneously activate both FXR and TGR5 signaling, and their nonoverlapping pathways, with potential additive effects.

Figure 8A is a bar graph showing relative albuminuria level defined by the ratio of albumin/creatinine (ACR) in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ- induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 8B are representative periodic acid-Schiff (PAS) staining images of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 50 µm.

Figure 8C is a bar graph showing glomerular area (µm ² ) as determined by periodic acid-Schiff (PAS) staining of kidney sections from nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 8D is a bar graph showing mesangial expansion index as determined by periodic acid-Schiff (PAS) staining of kidney sections from nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. Mesangial expansion index was defined by the percentage of mesangial area in glomerular tuft area, and the mesangial area was determined by assessment of PAS-positive and nucleus-free areas in the mesangium. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 8E are representative Masson trichrome staining images of kidney sections showing tubulointerstitial fibrosis (blue) from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom).

Figure 8F are representative merged two-photon excitation (green)-SHG (red) images of kidney sections showing tubulointerstitial fibrosis (red) from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 50 µm.

Figure 8G are immunofluorescence staining images for fibronectin of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 20 µm.

Figure 8H are immunofluorescence staining images for collagen IV of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 20 µm.

Figure 8I is a bar graph showing relative protein level of fibronectin in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 8J is a bar graph showing relative protein level of collagen IV in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 8K are immunohistochemical staining images for α-SMA of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom).

Figure 8L are immunohistochemical staining images for WT-1 of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 20 µm. Figure 8M is a bar graph showing relative podocyte density as numbers of podocytes per glomerular area in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 8N are immunofluorescence staining images for the podocyte marker nephrin of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ- induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom).

Figure 9A are oil red O staining images for neutral lipid accumulation of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom). Scale bar, 50 µm.

Figure 9B is a bar graph showing relative kidney triglyceride level (µmol/g) as determined by biochemical content analysis of kidney lipid extracts in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 9C is a bar graph showing relative kidney cholesterol level (µgl/g) as determined by biochemical content analysis of kidney lipid extracts in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 10A are immunofluorescence staining images for CD68 (red) and wheat-germ agglutinin (staining whole nephron; green) of kidney sections from nondiabetic DBA/2J mice (CON, top), DBA/2J mice with STZ-induced hyperglycemia (STZ, middle), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na (bottom).

Figure 10B is a bar graph showing relative mRNA level of p65 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON. Figure 10C is a bar graph showing relative mRNA level of p50 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 10D is a bar graph showing relative renal NF-κB transcriptional activation as determined by DNA binding assay in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 10E is a bar graph showing relative oxidative carbonylation of proteins in kidney homogenate as measured by ELISA in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 10F is a bar graph showing relative mRNA level of Nox-2 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 10G is a bar graph showing relative mRNA level of p22-phox in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 10H is a bar graph showing relative mRNA level of Nox-4 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 11A is a bar graph showing relative mRNA levels of HIF-1α in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 11B is a bar graph showing relative mRNA level of HIF-2α in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 11C is a Western blot showing relative protein level of Glut1 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na.

Figure 11D is a bar graph showing relative protein level of Glut1 in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 11E is a Western blot showing relative protein level of phospho-EIF-2α and total EIF-2α in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na

Figure 11F is a bar graph showing relative protein level of the ratio of phospho-EIF- 2α/EIF-2α in nondiabetic DBA/2J mice (CON), DBA/2J mice with STZ-induced

hyperglycemia (STZ), and DBA/2J mice with STZ-induced hyperglycemia treated with Compound 1-Na. ^† P < 0.05 versus STZ, *P < 0.05 versus CON.

Figure 12A is a bar graph showing relative protein level of FXR in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 12B is a bar graph showing relative protein level of TGR5 in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 12C are immunohistochemical staining images for FXR of kidney sections from nondiabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice treated with Compound 1-Na (bottom).

Figure 12D is a bar graph showing relative albuminuria protein level defined by the ratio of albumin/creatinine (ACR) in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 12E are periodic acid-Schiff (PAS) staining images of kidney sections from non-diabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice (db/db) treated with Compound 1-Na (bottom).

Figure 12F is a bar graph showing relative mesangial matrix index as determined by periodic acid-Schiff (pas) staining of kidney sections in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 12G are immunofluorescence microscopy staining images for podocyte marker synaptopodin of kidney sections from non-diabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice (db/db) treated with Compound 1-Na (bottom).

Figure 12H is a bar graph showing relative protein level of podocyte marker synaptopodin in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 12I are immunohistochemical staining images for type 1 collagen (collagen I) of kidney sections from nondiabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice treated with Compound 1-Na (bottom).

Figure 12J is a bar graph showing relative protein level of collagen I in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 12K are immunohistochemical staining images for type 3 collagen (collagen III) of kidney sections from nondiabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice treated with Compound 1-Na (bottom).

Figure 12L is a bar graph showing relative protein level of collagen III in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 12M are immunofluorescence microscopy staining images for fibronectin of kidney sections from non-diabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice (db/db) treated with Compound 1-Na (bottom). Figure 12N is a bar graph showing relative protein level of fibronectin in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 12O are immunofluorescence microscopy staining images for collagen IV of kidney sections from non-diabetic mature mice (db/m, top), diabetic mice (db/db, middle), and diabetic mice (db/db) treated with Compound 1-Na (bottom).

Figure 12P is a bar graph showing relative protein level of collagen IV in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 12Q is a bar graph showing relative urinary H ₂ O ₂ level in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 12R is a bar graph showing relative urinary thiobarbituric acid-reacting substances (TBARS) level in non-diabetic mature mice (db/m), diabetic mice (db/db), and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 13A is a Western blot showing relative protein level of phospho-AMPK and total AMPK in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na.

Figure 13B is a bar graph showing relative protein level of the ratio of phospho- AMPK/AMPK in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 13C is a Western blot showing relative protein level of SIRT-1 in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na.

Figure 13D is a bar graph showing relative protein level of the ratio of SIRT1/β-actin in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 13E is a Western blot showing relative protein level of PGC-1α in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na.

Figure 13F is a bar graph showing relative protein level of the ratio of PGC-1α/β-actin in non-diabetic mature mice (db/m), diabetic mice (db/db), non-diabetic mature mice (db/m) treated with Compound 1-Na, and diabetic mice (db/db) treated with Compound 1-Na. ^† P < 0.05 db/db + Compound 1-Na versus db/db, *P < 0.05 db/db versus db/m.

Figure 14A is a graph showing relative free NADH level in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with

Compound 1-Na as determined by label-free imaging with fluorescence lifetime imaging microscopy (FLIM).

Figure 14B is an exemplary phasor plot of free NADH (green cursor) and bound NADH (red cursor) with the line joining the two cursors as a metabolic trajectory.

Figure 14C is a bar graph showing relative mRNA level of proinflammatory TLR4 in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na. ^† P < 0.05 HF + Compound 1-Na versus LF, *P < 0.05 HF versus LF, **P < 0.05 HF + Compound 1-Na versus HF.

Figure 14D are label-free imaging (SHG-FLIM) images of kidney sections from low fat diet-fed mice (LF, top), high fat diet-fed mice (HF, middle), and high fat (HF) diet-fed mice treated with Compound 1-Na (bottom).

Figure 14E is a bar graph showing relative fibrosis level as determined by SHG and FLIM in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na. ^† P < 0.05 HF + Compound 1-Na versus LF, *P < 0.05 HF versus LF, **P < 0.05 HF + Compound 1-Na versus HF.

Figure 14F is a bar graph showing relative kidney ceramide level of total and major ceramide species accumulation in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). ^† P < 0.05 HF + Compound 1-Na versus LF, *P < 0.05 HF versus LF, **P < 0.05 HF + Compound 1-Na versus HF. Figure 14G is a bar graph showing relative kidney triglyceride level of total triglyceride species accumulation in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). ^† P < 0.05 HF + Compound 1-Na versus LF, *P < 0.05 HF versus LF, **P < 0.05 HF + Compound 1-Na versus HF.

Figure 14H is a bar graph showing relative kidney triglyceride level of major triglyceride species accumulation in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na as determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). ^† P < 0.05 HF + Compound 1-Na versus LF, *P < 0.05 HF versus LF, **P < 0.05 HF + Compound 1-Na versus HF.

Figure 15A is a bar graph showing relative bile acid level of total and major bile acid species composition in control low fat diet-fed mice (LF), high fat diet-fed mice (HF), and high fat (HF) diet-fed mice treated with Compound 1-Na. *P < 0.05 HF versus LF, **P < 0.05 HF + Compound 1-Na versus HF.

Figure 15B is a graph showing kidney bile acid composition in control low fat diet-fed mice (LF).

Figure 15C is a graph showing kidney bile acid composition in high fat diet-fed mice (HF).

Figure 15D is a graph showing kidney bile acid composition in high fat (HF) diet-fed mice treated with Compound 1-Na.

Figure 15E is a bar graph showing relative mRNA level of Cyp7b1 in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na. *P < 0.05 WD versus LF, **P < 0.05 WD + Compound 1-Na versus WD.

Figure 15F is a bar graph showing relative mRNA level of Cyp27a1 in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na. *P < 0.05 WD versus LF, **P < 0.05 WD + Compound 1-Na versus WD.

Figure 15G is a bar graph showing relative mRNA level of ASBT in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na. *P < 0.05 WD versus LF, **P < 0.05 WD + Compound 1-Na versus WD.

Figure 15H is a bar graph showing relative mRNA level of OSTα in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na. *P < 0.05 WD versus LF, **P < 0.05 WD + Compound 1-Na versus WD.

Figure 15I is a bar graph showing relative mRNA level of OSTβ in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na. *P < 0.05 WD versus LF, **P < 0.05 WD + Compound 1-Na versus WD.

Figure 15J is a bar graph showing relative mRNA level of MRP3 in control low fat diet-fed mice (LF), high fat Western diet-fed mice (WD), and high fat Western diet-fed mice (WD) treated with Compound 1-Na. *P < 0.05 WD versus LF, **P < 0.05 WD + Compound 1-Na versus WD.

Figure 15K is a schematic diagram illustrating bile acid synthesis mediated by Cyp27a1 and Cyp7b1 and bile transport from the urine mediated by bile acid transporter genes ASBT, OSTα, OSTβ, and MRP3. DETAILED DESCRIPTION

The present application is based at least in part on the discovery that a compound of Formula A or a pharmaceutically acceptable salt or amino acid conjugate thereof is effective in reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression, and reversing age-, diabetes-, and/or obesity-related impairments in mitochondrial biogenesis and mitochondrial function in a kidney.

Accordingly, the present application relates to a method of treating or preventing a renal disease, disorder, or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A:

(A),

or a pharmaceutically acceptable salt or amino acid conjugate thereof, wherein:

R ₁ is C ₁ -C ₆ alkyl;

R2, R3, and R5 are each independently H or OH; and

In one embodiment, the renal disease, disorder, or condition is a renal disease, disorder, or condition that is modulated by FXR (e.g., where the expression of FXR plays a role in the initiation and/or development of the renal disease, disorder, or condition). In one embodiment, the renal disease, disorder, or condition is a renal disease, disorder, or condition that is modulated by TGR5 (e.g., where the expression of TGR5 plays a role in the initiation and/or development of the renal disease, disorder, or condition). In one embodiment, the renal disease, disorder, or condition is a renal disease, disorder, or condition that is modulated by FXR and TGR5. In one embodiment, the renal disease, disorder, or condition is an age-, diabetes-, and/or obesity-related renal disease, disorder, or condition. In one embodiment, the age-, diabetes-, and/or obesity-related renal disease, disorder, or condition is modulated by FXR. In one embodiment, the age-, diabetes-, and/or obesity-related renal disease, disorder, or condition is modulated by TGR5. In one embodiment, the age-, diabetes-, and/or obesity- related renal disease, disorder, or condition is modulated by FXR and TGR5. In one embodiment, the renal disease, disorder, or condition or age-, diabetes-, and/or obesity-related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of FXR. In one embodiment, the renal disease, disorder, or condition or age-, diabetes-, and/or obesity-related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of TGR5. In one embodiment, the renal disease, disorder, or condition or age-, diabetes-, and/or obesity- related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of FXR and TGR5. In one embodiment, the renal disease, disorder, or condition is a diabetes- and/or obesity-related renal disease, disorder, or condition. In one embodiment, the diabetes- and/or obesity-related renal disease, disorder, or condition is modulated by FXR. In one

embodiment, the diabetes- and/or obesity-related renal disease, disorder, or condition is modulated by TGR5. In one embodiment, the diabetes- and/or obesity-related renal disease, disorder, or condition is modulated by FXR and TGR5. In one embodiment, the renal disease, disorder, or condition or diabetes- and/or obesity-related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of FXR. In one embodiment, the renal disease, disorder, or condition or diabetes- and/or obesity- related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of TGR5. In one embodiment, the renal disease, disorder, or condition or diabetes- and/or obesity-related renal disease, disorder, or condition is a renal disease, disorder, or condition that is associated with decreased expression of FXR and TGR5.

In one embodiment, an age-related renal disease, disorder, or condition is a renal disease, disorder, or condition that starts to develop in a subject in a particular age group. In one embodiment, the subject is between 45 and 95 years of age, between 50 and 95 years of age, between 55 and 95 years of age, between 60 and 95 years of age, between 65 and 95 years of age, between 45 and 85 years of age, between 50 and 85 years of age, between 55 and 85 years of age, between 60 and 85 years of age, between 65 and 85 years of age, between 45 and 75 years of age, between 50 and 75 years of age, between 55 and 75 years of age, between 60 and 75 years of age, or between 65 and 75 years of age.

In one embodiment, the renal disease, disorder, or condition is selected from renal inflammation, renal oxidative stress, renal lipid accumulation, renal fibrosis, renovascular diseases (e.g., narrowing or blockage of the renal artery caused by deposits of fatty acids, cholesterol, calcium, and/or other substances in the renal arteries), diabetic nephropathy, and chronic kidney disease.

The present application also relates to a method of improving one or more kidney functions in a subject in need thereof, comprising administering to the subject a

therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof. In one embodiment, the kidney functions include regulation of extracellular fluid volume, for example, to ensure an adequate quantity of plasma to keep blood flowing to vital organs, regulation of the osmolarity of extracellular fluid, maintaining ion concentration (e.g., sodium ions, potassium ions, and calcium ions), maintaining the pH of the blood plasma, excretion of wastes and toxins into urine, producing hormones or red blood cells, maintaining bone health, and controlling blood pressure.

The present application also relates to a method of regulating one or more

complementary or nonoverlapping signaling and/or metabolic pathways involved in nephropathy. In certain embodiments, the regulating includes, but is not limited to, improving proteinuria, preventing podocyte injury, preventing mesangial expansion, preventing tubulointerstitial fibrosis, inhibiting endoplasmic reticulum stress, inhibiting enhanced renal fatty acid and cholesterol metabolism, preventing mitochondrial dysfunction, preventing oxidative stress, preventing kidney fibrosis, and/or stimulating a signaling cascade involving, for example, AMP-activated protein kinase, sirtuin 1, PGC-1α, sirtuin 3, estrogen-related receptor-α, and/or Nrf-1.

In one embodiment, one or more kidney functions, such as the functions described herein above, are improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after the administration of a compound of the present application. In one embodiment, one or more kidney functions in the subject are impaired (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the normal level of the one or more kidney functions), and improving one or more kidney functions by administration of a compound of the present application restores the one or more kidney functions to 50%, 60%, 70%, 80%, 90%, or 100% of the normal level of the one or more kidney functions before the impairment. In one embodiment, one or more kidney functions in the subject are decreased as compared to a control subject (e.g., a control subject as described herein). In one embodiment, one or more kidney functions in the subject are decreased (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the level of the one or more kidney functions in a control subject), and improving one or more kidney functions by administration of a compound of the present application restores the one or more kidney functions to 50%, 60%, 70%, 80%, 90%, or 100% of the level of the one or more kidney functions in the control subject. The present application also relates to a method of slowing the progress of or reversing age-, diabetes-, and/or obesity-related increase in proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression in a kidney, or slowing the progress of or reversing age-, diabetes-, and/or obesity- related impairments in mitochondrial biogenesis or mitochondrial function in a kidney, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression in a kidney is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after the

administration of a compound of the present application. In one embodiment, proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression in a kidney is increased in the subject (e.g., to 120%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, or 400% of the normal level), and by administration of a compound of the present application, proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression is decreased to 150%, 140%, 130%, 120%, 100%, or 90% of the normal level. In one embodiment, proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression in the subject is increased as compared to a control subject (e.g., a control subject as described herein). In one embodiment, proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression in the subject is increased (e.g., to 120%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, or 400% of the level in a control subject), and administration of a compound of the present application decreases proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression to 150%, 140%, 130%, 120%, 100%, or 90% of the level in the control subject.

The level of proteinuria, podocyte injury, fibronectin and/or type 4 collagen accumulation in the glomeruli of a kidney, triglyceride and/or cholesterol accumulation in the glomeruli and/or tubulointerstitium of a kidney, or TGF-β expression can be determined by methods and materials known in the art. For example, the level of proteinuria can be determined by measuring the amount of albuminuria or the ratio of albuminuria to creatinine. In another example, the level of fibronectin accumulation or TGF-β expression can be determined either by measuring the amount of mRNA or protein of fibronectin or TGF-β.

Podocyte injury can be manifested in many ways. Abnormal podocyte death (e.g., through programmed cell death, also known as apoptosis), detachment of podocytes from glomerular basement membrane (GBM), lack of podocyte proliferation, and/or abnormal podocyte mitosis are several indications of injuries to podocytes in a kidney. These abnormalities can be identified by various methods known in the art. For example, apoptotic or detached podocytes can be detected in urine through immunostaining with podocyte- specific antibodies, such as nephrin, podocin, and Glepp-1. In addition, irregular proliferation and mitosis of podocytes can be assessed with cultured podocyte in vitro.

In one embodiment, the level of mitochondrial biogenesis or mitochondrial function is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after the administration of a compound of the present application. In one embodiment, the level of mitochondrial biogenesis or mitochondrial function is decreased in a kidney in the subject (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the normal level of mitochondrial biogenesis or mitochondrial function), and increasing the level of mitochondrial biogenesis or mitochondrial function by administration of a compound of the present application restores the level of mitochondrial biogenesis or mitochondrial function to 50%, 60%, 70%, 80%, 90%, or 100% of the normal level of mitochondrial biogenesis or mitochondrial function before the decrease. In one embodiment, the level of mitochondrial biogenesis or

mitochondrial function in the subject is decreased as compared to a control subject (e.g., a control subject as described herein). In one embodiment, the level of mitochondrial biogenesis or mitochondrial function in the subject is decreased (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the level of mitochondrial biogenesis or mitochondrial function in a control subject), and increasing the level of mitochondrial biogenesis or mitochondrial function by administration of a compound of the present application restores the level of mitochondrial biogenesis or mitochondrial function to 50%, 60%, 70%, 80%, 90%, or 100% of the level of mitochondrial biogenesis or mitochondrial function in the control subject.

Methods to measure mitochondrial biogenesis or mitochondrial function are known in the art. For example, the activity of mitochondrial complex I or complex IV can be determined through measurement of NADH dehydrogenase or cytochrome C oxidase activity in a cell. In addition, mitochondrial biogenesis can be assessed by measuring the ratio of the amount of mitochondria DNA to the amount of nuclear DNA in a cell.

The present application also relates to a method of increasing the level of FXR and/or TGR5 in a kidney in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula A, or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, the level of FXR and/or TGR5 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after the administration of a compound of the present application. In one embodiment, the level of FXR and/or TGR5 is decreased in a kidney in the subject (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the normal level of FXR and/or TGR5), and increasing the level of FXR and/or TGR5 by administration of a compound of the present application restores the level of FXR and/or TGR5 to 50%, 60%, 70%, 80%, 90%, or 100% of the normal level of FXR and/or TGR5 before the decrease. In one embodiment, the level of FXR and/or TGR5 in the subject is decreased as compared to a control subject (e.g., a control subject as described herein). In one embodiment, the level of FXR and/or TGR5 in the subject is decreased (e.g., to 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the level of FXR and/or TGR5 in a control subject), and increasing the level of FXR and/or TGR5 by administration of a compound of the present application restores the level of FXR and/or TGR5 to 50%, 60%, 70%, 80%, 90%, or 100% of the level of FXR and/or TGR5 in the control subject.

In one embodiment, the methods of the present application further comprise measuring the level of one or more targets in the subject or a cell from a kidney of the subject, wherein the target is selected from FXR, TGR5, synaptopodin, Nrf-1, pAMPK, Sirt1, Sirt3, ERRα, PGC1α, MCAD, Cox4, LCAD, TGF-β, fibronectin, FSP-1, TNF-α, TLR2, TLR4, and acetyl- IDH2.

In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the level of one or more targets is decreased in the subject as compared to the normal level of the one or more targets in the subject or as compared to a control subject. In one embodiment, the target is selected from CD36, LPL, FXR, TGR5, synaptopodin, Nrf-1, pAMPK, Sirt1, Sirt3, ERRα, PGC1α, MCAD, Cox4, and LCAD. In one embodiment, the target is involved in mitochondrial biogenesis, including Nampt (nicotinamide phosphoribosyl transferase), Sirt1, Sirt3, PGC-1α, ERR-α, Nrf1, and LCAD (long-chain acyl CoA

dehydrogenase). In one embodiment, the target is a M2 macrophage marker, including CD163 and CD206.

In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the level of one or more targets is increased in the subject as compared to the normal level of the one or more targets in the subject or as compared to a control subject. In one embodiment, the target is selected from SREBP-1, SCD-1, SCD-2, Fit-1, kidney triglyceride, kidney cholesterol, p65, p50, NFκB, Nox-2, Nox-4, Hif1a, Hif2a, Glut1, p- EIF2α, collagen I, collagen III, collagen IV, p22-phox, CD68, ICAM-1, Cox2, CTGF, FSP-1, Snail, ZEB1, TGF-β, fibronectin, FSP-1, acetyl-IDH2, TNF-α, TLR2, and TLR4.

In one embodiment, the methods of the present application further comprise measuring the ratio of the amount of mitochondria DNA to the amount of nuclear DNA in a cell from a kidney of the subject. In one embodiment, the methods of the present application further comprise measuring the ratio of the amount of pAMPK to the amount of AMPK in a cell from a kidney of the subject. In one embodiment, the methods of the present application further comprise measuring the ratio of the amount of acetyl-IDH2 to the amount of IDH2 in a cell from a kidney of the subject.

In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the ratio of the amount of mitochondria DNA to the amount of nuclear DNA is decreased in the subject as compared to the normal ratio in the subject or as compared to a control subject. In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the ratio of the amount of pAMPK to the amount of nuclear AMPK is decreased in the subject as compared to the normal ratio in the subject or as compared to a control subject. In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the ratio of the amount of acetyl-IDH2 to the amount of IDH2 is increased in the subject as compared to the normal ratio in the subject or as compared to a control subject.

In one embodiment, the methods of the present application further comprise measuring the amount of albuminuria or the ratio of the amount of albuminuria to the amount of creatinine in the subject. In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the amount of albuminuria or the ratio of the amount of albuminuria to the amount of creatinine is increased in the subject as compared to the normal amount of albuminuria or normal ratio of the amount of albuminuria to the amount of creatinine in the subject, or as compared to a control subject.

In one embodiment, the methods of the present application further comprise measuring the activity of mitochondrial complex I and/or complex IV in the subject. In one embodiment, the methods of present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, when the activity of mitochondrial complex I and/or complex IV is decreased as compared to the normal activity of mitochondrial complex I and/or complex IV in the subject or as compared to a control subject.

In one embodiment, the methods of the present application comprise administering to a subject in need thereof a therapeutically effective amount of a compound of the present application, in combination with reducing the calorie uptake in the subject. In one

embodiment, reducing calorie uptake comprises administering to the subject a diet that has a reduced amount of calorie. In one embodiment, the amount of calorie is reduced by 10%, by 20%, by 30%, by 40%, by 50%, by 60%, or by 70% as compared to the normal calorie uptake by the subject.

In one embodiment, the subject is a human. In one embodiment, the subject is a human over 45 years of age, over 50 years of age, over 55 years of age, over 60 years of age, over 65 years of age, or over 70 years of age. In one embodiment, the subject is a human over 65 years of age. In one embodiment, the subject is a human between 45 and 95 years of age, between 50 and 95 years of age, between 55 and 95 years of age, between 60 and 95 years of age, between 65 and 95 years of age, between 45 and 85 years of age, between 50 and 85 years of age, between 55 and 85 years of age, between 60 and 85 years of age, between 65 and 85 years of age, between 45 and 75 years of age, between 50 and 75 years of age, between 55 and 75 years of age, between 60 and 75 years of age, or between 65 and 75 years of age.

In one embodiment, the control subject is a human. In one embodiment, the control subject is a human less than 70 years of age, less than 65 years of age, less than 60 years of age, less than 55 years of age, less than 50 years of age, or less than 45 years of age. In one embodiment, the control subject is a human less than 65 years of age.

In one embodiment, the subject has one or more diseases, disorders, or conditions in addition to the renal disease, disorder, or condition. In one embodiment, the additional disease, disorder, or condition is selected from a cardiovascular disease, hypertension, a metabolic syndrome, diabetes, obesity, and insulin resistance.

Compounds and compositions of the application can be administered in therapeutically effective amounts in a combinatorial therapy with one or more therapeutic agents

(pharmaceutical combinations) or modalities. Where the compounds of the application are administered in conjunction with other therapies, dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth. For example, synergistic effects can occur with substances.

Combination therapy includes the administration of the subject compounds in further combination with one or more other biologically active ingredients (such as, but not limited to, a FXR agonist, a TGR5 agonist, a second compound of Formula A, a second and different compound of Formula A) and non-drug therapies (such as, but not limited to, surgery or dietary treatment). For instance, the compounds of the application can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the application. The compounds of the application can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy or treatment modality. In general, a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy.

In another aspect of the application, the compounds may be administered in combination with one or more separate pharmaceutical agents, e.g., a chemotherapeutic agent, an immunotherapeutic agent, or an adjunctive therapeutic agent.

In one embodiment the compound is a compound of Formula A:

(A),or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, R ₁ is C ₁ -C ₆ alkyl selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, and hexyl. In one embodiment, R1 is C1-C4 alkyl selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, and t-butyl. In one embodiment, R1 is methyl, ethyl, n-propyl, or i-propyl. In one embodiment, R ₁ is methyl or ethyl. In one embodiment, R ₁ is methyl. In one embodiment, R ₁ is ethyl.

In one embodiment, R2 is H and R3 is OH. In one embodiment, R3 is H and R2 is OH. In one embodiment, R ₅ is H. In one embodiment, R ₅ is OH.

In one embodiment, R ₂ is H, R ₃ is OH, and R ₅ is H. In one embodiment, R ₂ is H, R ₃ is OH, and R5 is OH.

In one embodiment, R4 is CO2H. In one embodiment, R4 is OSO3H.

In one embodiment, R ₂ is H, R ₃ is OH, R ₄ is CO ₂ H, and R ₅ is H. In one embodiment, R2 is H, R3 is OH, R4 is OSO3H, and R5 is OH.

In one embodiment, R1 is ethyl, R2 is H, R3 is OH, R4 is CO2H, and R5 is H. In one embodiment, R ₁ is ethyl, R ₂ is H, R ₃ is OH, R ₄ is OSO ₃ H, and R ₅ is H.

In one embodiment, a compound of Formula A is Compound 1:

or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, a com ound of Formula A is Com ound 2:

2),

or a pharmaceutically acceptable salt or amino acid conjugate thereof.

In one embodiment, a pharmaceutically acceptable salt of Compound 1 is the sodium salt of Compound 1 (i.e., Compound 1-Na). In yet another embodiment, a pharmaceutically acceptable salt of Compound 1 is the triethylammonium salt of Compound 1 (i.e., Compound 1-TEA).

In one embodiment, a compound of the present application is administered once daily, twice daily, three times daily, once every 6 hours, or once every 4 hours. In one embodiment, a compound of the present application is administered for one day, two days, three days, four days, five days, six days, or seven days a week. In one embodiment, a compound of the present application is not administered every day of the week. In one embodiment, a compound of the present application is administered every other day, once every three days, once every four days, once every five days, once every six days, or once every seven days.

In one embodiment, a compound of the present application is administered for a period of one week, two weeks, three weeks, four weeks, six weeks, two months, three months, four months, six months, or more. In one embodiment, the period in which a compound of the present application is administered comprises one or more segments (e.g., one or more days, one or more weeks, or one or more months) during which the compound is not administered. In one embodiment, the one or more segments during which the compound is not

administered are preceded by and followed by administration of the compound. As used herein, the term“Compound 1” refers to

which is also known as 6α-ethyl-3α,7α,23-trihydroxy-24-nor-5β-cholan-23-hydroge n sulphate.“Compound 1-Na” or“1-Na” which is also known as INT-767 or 6α-ethyl- 3α,7α,23-trihydroxy-24-nor-5β-cholan-23-sulphate sodium" are used interchangeably, and refer to the sodium salt of Compound 1. As used herein,“Compound 1-TEA” or“1-TEA” is used interchangeably, and refer to the triethylammonium salt of Compound 1. The structures of Compound 1-Na and Compound 1-TEA are provided below.

The phrase a“compound of the application” or“compound of the present application” as used herein encompasses Compound 1, 1-Na, 1-TEA, or a pharmaceutically acceptable salt or amino acid conjugate thereof.

As used herein, the term“amino acid conjugate” refers to a conjugate of the compound of the present application with any suitable amino acid. For example, such a suitable amino acid conjugate of a compound of the present application will have the added advantage of enhanced integrity in bile or intestinal fluids. Suitable amino acids include but are not limited to glycine and taurine. Thus, the present invention encompasses the glycine and taurine conjugates of the compound of the present application (e.g., Compound 1).

As used herein, FXR refers to Farnesoid X Receptor, which is a member of the nuclear receptor family of ligand-activated transcription factors that includes receptors for the steroid, retinoid, and thyroid hormones. FXR binds to DNA as a heterodimer with the 9-cis retinoic acid receptor (RXR). As used herein, TGR5 refers to a G-protein-coupled receptor that is responsive to bile acids (BAs).

As used herein, a“subject in need thereof” is a subject having a disease, disorder, or condition against which a compound of the application is effective, or a subject having an increased risk of developing a disease, disorder, or condition against which a compound of the application is effective relative to the population at large. A“subject” includes a mammal. The mammal can be any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow, horse, goat, camel, sheep or a pig. Particularly, the mammal is a human.

The term“treating” as used herein refers to any indicia of success in the treatment or amelioration of any of the diseases, disorders, or conditions described herein. Treating can include, for example, reducing or alleviating the severity of one or more symptoms of any of the diseases, disorders, or conditions described herein, or it can include reducing the frequency with which symptoms of any of the diseases, disorders, or conditions described herein are experienced by a patient.“Treating” can also refer to reducing or eliminating any of the diseases, disorders, or conditions described herein of a part of the body, such as a cell, tissue or bodily fluid, e.g., podocytes.

As used herein, the term“preventing” refers to the partial or complete prevention of any of the diseases, disorders, or conditions described herein in an individual or in a population, or in a part of the body, such as a cell, tissue or bodily fluid (e.g., podocytes). The term“prevention” does not establish a requirement for complete prevention of a disease, disorder, or condition in the entirety of the treated population of individuals or cells, tissues, or fluids of individuals.

The term“treat or prevent” is used herein to refer to a method that results in some level of treatment or amelioration of any of the diseases, disorders, or conditions described herein, and contemplates a range of results directed to that end, including but not restricted to prevention of any of the diseases, disorders, or conditions described herein entirely.

Unless specified or the context dictates otherwise,“renal” and“kidney” are used interchangeably.

As used herein,“pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

A“pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A“pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient.

The phrase“therapeutically effective amount” as used herein refers to an effective amount comprising an amount sufficient to treat a disease, disorder, or condition described herein or to prevent or delay a disease, disorder, or condition described herein. In some embodiments, an effective amount is an amount sufficient to delay the development of the disease, disorder, or condition. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations.

A therapeutically effective amount can be estimated initially either in cell culture assays, e.g., in a cell from the kidney, such as podocytes, or animal models, usually rats, mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic/prophylactic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio,

LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred.

The term“regimen” as used herein refers to a protocol for dosing and/or timing the administration a compound of the application. A regimen can include periods of active administration and periods of rest as known in the art. Active administration periods include administration of a compound of the application in a defined course of time, including, for example, the number of and timing of dosages of the compositions. In some regimens, one or more rest periods can be included where no compound is actively administered, and in certain instances, includes time periods where the efficacy of such compounds can be minimal.

As used herein,“combination therapy” means that a compound of the application can be administered in conjunction with another therapeutic agent.“In conjunction with” refers to administration of one treatment modality in addition to another treatment modality, such as administration of a compound of the application as described herein in addition to

administration of another therapeutic agent to the same subject. As such,“in conjunction with” refers to administration of one treatment modality before, during, or after delivery of a second treatment modality to the subject.

The term“about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some embodiments ±5%, in some embodiments ±1%, and in some embodiments ±0.1% from the specified value, as such variations are appropriate to practice the disclosed methods or to make and used the disclosed compounds and in the claimed methods.

Unless specified or the context dictates otherwise, a“pharmaceutical composition” or “pharmaceutical formulation” is used interchangeably, and refers to a formulation containing a compound of the present application in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. It can be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. The specification for the dosage unit forms is dictated by and directly dependent on the unique characteristics of the active reagent and the particular therapeutic effect to be achieved. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial.

Possible formulations include those suitable for oral, sublingual, buccal, parenteral (e.g., subcutaneous, intramuscular, or intravenous), rectal, topical including transdermal, intranasal, and inhalation administration. Most suitable means of administration for a particular patient will depend on the nature and severity of the disease being treated, the nature of the therapy being used, and the nature of the active compound.

Formulations suitable for oral administration may be provided as discrete units, such as tablets, capsules, cachets, lozenges, each containing a predetermined amount of the active compound; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water or water-in-oil emulsions.

Formulations suitable for sublingual or buccal administration include lozenges comprising a compound of the application and typically a flavored base, such as sugar and acacia or tragacanth and pastilles comprising the active compound in an inert base, such as gelatin and glycerin or sucrose acacia.

Formulations suitable for parenteral administration typically comprise sterile aqueous solutions containing a predetermined concentration of the active compound; the solution may be isotonic with the blood of the intended recipient. Additional formulations suitable for parenteral administration include formulations containing physiologically suitable co-solvents and/or complexing agents such as surfactants and cyclodextrins. Oil-in-water emulsions are also suitable formulations for parenteral formulations. Although such solutions may be administered intravenously, they may also be administered by subcutaneous or intramuscular injection.

Formulations suitable for rectal administration may be provided as unit-dose suppositories comprising a compound of the application in one or more solid carriers forming the suppository base, for example, cocoa butter.

Formulations suitable for topical or intranasal application include ointments, creams, lotions, pastes, gels, sprays, aerosols, and oils. Suitable carriers for such formulations include petroleum jelly, lanolin, polyethyleneglycols, alcohols, and combinations thereof.

Oral formulations generally include an inert diluent or an edible pharmaceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral administration, the active ingredient can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral formulations can also be prepared using a fluid carrier for use as a mouthwash, wherein the active ingredient in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes®; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Pharmaceutically compatible diluents may also include starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, and the like. Pharmaceutically compatible wetting agents included water, ethanol, isopropanol, and the like. Pharmaceutically compatible binders may also include starch pulp, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, mucilage of arabic gum, gelatin mucilage, sodium hydroxymethylcellulose, methylcellulose,

hydroxypropylmethylcellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, and the like. Pharmaceutically compatible disintegrants may also include dry starch, microcrystalline cellulose, low-substituted hydroxypropylcellulose, cross-linked polyvinylpyrrolidone, croscarmellose sodium, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid esters, sodium dodecyl sulfonate and the like. Pharmaceutically compatible lubricants and glidants may also include talc powder, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.

Pharmaceutical formulations suitable for injectable use (e.g., intravenous,

intramuscular) include sterile aqueous solutions (where water soluble),

dispersions/suspensions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Suitable carriers include physiological saline,

bacteriostatic water, Cremophor EL ^ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carriers can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against contaminating by microorganisms such as bacteria and fungi. The proper fluidity can be maintained, for example, by the use of agents such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of

microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Other excipients include, but are not limited to, antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. The preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Sterile injectable solutions can be prepared by incorporating the active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Formulations of the application may be prepared by any suitable method, typically by uniformly and intimately admixing a compound of the application with liquids or finely divided solid carriers or both, in the required proportions and then, if necessary, shaping the resulting mixture into the desired shape.

For example, a tablet may be prepared by compressing an intimate mixture comprising a powder or granules of the active ingredient and one or more optional ingredients, such as a binder, lubricant, inert diluent, or surface active dispersing agent, or by molding an intimate mixture of powdered active ingredient and inert liquid diluent. Suitable formulations for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers, or insufflators.

In addition to the ingredients specifically mentioned above, the formulations of the present application may include other agents known to those skilled in the art of pharmacy, having regard for the type of formulation in issue. For example, formulations suitable for oral administration may include flavoring agents and formulations suitable for intranasal administration may include perfumes. Dosage and administration may be adjusted to provide sufficient levels of the active agent(s) (e.g., a compound of the present application) to maintain a desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Pharmaceutical compositions may be administered every 1-96 hours, preferably every 2-72 hours, preferably every 3-48 hours, preferably every 4-24 hours, preferably every 6-12 hours. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of from about 0.1- 1500 mg, 0.2-1200 mg, 0.3-1000 mg, 0.4-800 mg, 0.5-600 mg, or 0.6-500 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of from 0.5-20 mg, preferably 1-15 mg, preferably 2-14 mg, preferably 4-12 mg, preferably 5-10 mg.

In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 50 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 40 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 30 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 20 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 15 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 10 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 5 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 2.5 mg. In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of about 1 mg.

In one embodiment, a pharmaceutical composition is administered in a dosage form which comprises a compound of the application in a daily total amount of less than 10 mg/kg, preferably less than 5 mg/kg, such as, for example 0.1-5.0 mg/kg, preferably 0.5-4.5 mg/kg, preferably 1.0-4.0 mg/kg, preferably 1.2-3.5 mg/kg, preferably 1.4-3.0 mg/kg, preferably 1.5- 2.5 mg/kg, preferably 1.6-2.4 mg/kg. Example 1. Synthesis of a compound of the present application

A compound of the present application can be prepared by methods known in the art (e.g., those described in U.S. Patent No.7,932,244). For example, a compound of the present application can be prepared by a process as shown in Scheme 1 and disclosed in WO

2014/066819.

Step 1 is the esterification of Compound 2 to obtain Compound 4. Step 2 is a reaction to form Compound 5 from Compound 4. Step 3 is the protection of the hydroxy group at the C3 position of Compound 5 to afford Compound 6. Step 4 is the oxidative cleavage of Compound 6 to afford Compound 7. Step 5 is the reduction of Compound 7 to afford Compound 8. Step 6 is the sulfonation of Compound 8 to afford the sodium salt of

Compound 1 (1-Na). The sodium salt of Compound 1 can be converted to its free acid form (i.e., Compound 1) or other salt forms (e.g., Compound 1-TEA or the triethylammonium salt of Compound 1) according to procedures known in the art. Example 2. Methods of the present application

^ Animal models

1) Aging mice: 3-month-old and 22-month-old C57BL/6 mice fed ad lib, and 22- month-old C57BL/6 mice with caloric restriction were obtained through the NIA aging colony. The mice were continued to be fed ad lib with NIH31 diet or caloric restricted NIH31 diet as per the NIA instructions. A group of 22-month-old ad lib fed mice were also fed with diet containing Compound 1-Na (6α-ethyl-3α, 7α, 23-trihydroxy-24-nor-5-β-cholan-23 sulfate sodium salt) at 30 mg/kg body weight/day.

2) Ames Mice: Ames dwarf mice and their controls were acquired from Jackson Laboratories (Bar Harbor, ME) and studied at 21-month-old.

3) DBA/2J Mice: Eight-week-old male DBA/2J mice were obtained from the Jackson Laboratories (Bar Harbor, ME). They were maintained on a 12-hour light/12-hour dark cycle. Mice were injected with streptozotocin (STZ) (Sigma-Aldrich, St. Louis, MO)

intraperitoneally (40 mg/kg in 50 mM sodium citrate buffer, pH 4.5) for 5 consecutive days or 50 mM sodium citrate solution only. Tail vein blood glucose levels were measured 1 week after the last STZ injection, and mice with glucose levels > 250 mg/dl were considered diabetic. DBA/2J mice were fed with a Western diet (21% milkfat, 0.15% cholesterol, TD88137; Harlan-Teklad, Madison, WI). Mice were treated for 8 weeks with (i) Western diet only; (ii) Compound 1-Na (6α-ethyl-3α, 7α, 23-trihydroxy-24-nor-5-β-cholan-23 sulfate sodium salt): 30 mg/kg body weight/day admixed with Western diet; (iii) Compound 2 (obeticholic acid): 20 mg/kg body wt/d admixed with Western diet; or (iv) Compound 3: 30 mg/kg body wt/d admixed with Western diet.

4) db/db Mice: Six-week-old male db/m and db/db mice (BLKS/J) were obtained from the Jackson Laboratories. They were maintained on a 12-hour light/12-hour dark cycle. They were on (i) a regular chow diet, (ii) Compound 1-Na (6α-ethyl-3α, 7α, 23-trihydroxy-24- nor-5-β-cholan-23 sulfate sodium salt) (30 mg/kg body wt/d), (iii) Compound 2 (obeticholic acid) (20 mg/kg body wt/d), or (iv) Compound 3 (30 mg/kg body wt/d) admixed with chow for 2 weeks.

5) Diet-Induced Obesity Mice: Six-week-old male C57BL/6J mice were obtained from the Jackson Laboratories. They were maintained on a 12-hour light/12-hour dark cycle. They were fed a matched (i) control diet (10 kcal% fat), (ii) a high-fat diet (60 kcal% fat; Research Diets), or (iii) a high-fat diet containing Compound 1-Na (6α-ethyl-3α, 7α, 23- trihydroxy-24-nor-5-β-cholan-23 sulfate sodium salt): 30 mg/kg body wt/d admixed in the diet.

^ Blood and Urine chemistry

Blood glucose levels were measured with a Glucometer (Elite XL; Bayer, Tarrytown, NY). Plasma lipid levels were measured with kits (Wako Chemical, Richmond, VA). Urine albumin and creatinine concentrations were determined with kits (Exocell, Philadelphia, PA) according to the manufacturer’s manual.

^ RNA extraction and quantitative real-time PCR

Approximately 300-500 ng RNA was used to generate barcoded RNA libraries using Lexogen’s Quant-sEquation 3’ mRNA seq kit modified per the manufacturer’s

recommendations for low input/partial degradation (Lexogen, Vienna, Austria). ERCC controls were spiked in at the manufacturer’s recommended concentrations (Life

Technologies, Carlsbad, CA). Library quantification and quality control were performed using the High Sensitivity DNA Kit and the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). Precise library quantification was performed using the Ion Library Quantitation Kit (Life Technologies), which uses Taqman qPCR chemistry to quantify adapter ligated library fragments; these results were used to determine library input into the template reaction, and templates for sequencing were prepared using the 200-bp v3 OT2 kit and the Ion One Touch 2 platform (Life Technologies) with sequencing performed on an Ion Proton with signal processing and base calling using Ion Torrent Suite, version 5.0.4 (Life Technologies). Raw sequence was mapped to Ampliseq-supported mm10 transcriptome. Quality control metrics and normalized read counts per million were generated using the RNA-Seq Analysis plugin (v.5.0.3.0; Ion Torrent community; Life Technologies).

Quantitative real-time PCR was performed according to methods known in the art, for example, as in Jiang T. et al., Diabetes 56, 2485 (2007), Wang XX. et al., Diabetes 59, 2916 (2010), Wang XX. et al., Am. J. Physiol. Renal Physiol.297, F1587 (2009), or Wang XX. et al., J. Am. Soc. Nephrol.27, 1362 (2016).

^ Western blotting

Cortical homogenate protein content was measured by BCA assay (Thermo Fisher Scientific, Waltham, MA). Equal amount of total protein was separated by SDS-PAGE gels and transferred onto PVDF membranes. The antibodies against SREBP-1 (catalog no. H-160; Santa Cruz Biotechnology, Dallas, TX), Glut1 (catalog no.07-1401; Cell Signaling, Danvers, MA), p-AMPK/AMPK (catalog nos.4184 and 2795; Cell Signaling), SIRT1 (catalog no.07- 131; Millipore, Billerica, MA), PGC-1α (catalog no. AB3242; Millipore), pEIF-2α/EIF-2α (catalog nos.9721 and 9722; Millipore), and SIRT3 (catalogue number 5490, Cell Signaling) were used for Western blotting. After HRP-conjugated secondary antibodies, the immune complexes were detected by chemiluminescence captured on UVP Biospectrum 500 Imaging System (Upland, CA) and the densitometry was performed with ImageJ software. β-actin (catalogue number A5316, Sigma, St Louis, MO) was used as a loading control and all signals were normalized to β-actin signal.

^ Lipid Extraction and Measurement of Lipid Composition

Lipids from the kidneys were extracted by the method of Bligh and Dyer. Triglyceride and cholesterol composition were measured by gas chromatography (Agilent Technologies, Wilmington, DE).

^ Histology staining and Immunofluorescence microscopy

Sections (4-µm-thick) cut from 10% formalin-fixed, paraffin-embedded kidney samples were used for periodic acid-Schiff staining and Masson trichrome staining. Frozen sections were used for oil red O staining of neutral lipid deposits or immunostaining for nephrin (provided by Larry Holzman, University of Pennsylvania, Philadelphia, PA), synaptopodin (Sigma-Aldrich), fibronectin (Sigma-Aldrich), CD68 (AbD Serotec, Raleigh, NC), and α-SMA (Sigma-Aldrich) and imaged with a laser-scanning confocal microscope (LSM 510; Zeiss, Jena, Germany). The expression level was quantified as sum of pixel values per glomerular area using ImageJ (version 1.44) image analysis software.

Frozen sections (4μm-thick) were used for immunostaining for synaptopodin

(catalogue number S9442, Sigma) and fibronectin (catalogue number F3648, Sigma), and imaged with a laser scanning confocal microscope (LSM 780, Zeiss, Germany).

^ Mitochondrial complex activity assay

Mitochondrial fraction was isolated from the kidney and used for the measurement of complex I (NADH dehydrogenase) and complex IV (cytochrome c oxidase) activity with kits from Abcam (Cambridge, UK).

^ Podocyte cell culture Human podocytes obtained were maintained in RPMI-1640, 1% Insulin-Transferrin- Selenium, 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 33 °C. Podocyte differentiation is induced by thermo-shifting the cells from 33°C to 37°C for 7 days. The differentiated podocytes were then cultured in the presence of 5% of the 4-month old C57/BL6 mouse serum or 5% of the 28-month old mouse serum obtained from NIA to replace fetal bovine serum for 72 hours. In the last 24 hours, 10µM Compound I-Na was added to the treatment group.

^ Liquid Chromatography-Tandem Mass Spectrometry Techniques for Ceramide Species, Triglyceride Species, and Bile Acid Composition

1) For Kidney Ceramide and Triglyceride Composition:

Kidney tissue was homogenized with 500 µL methanol:H2O (4:3, vol/vol) solution and then extracted using 700 µL chloroform containing SM (17:0), PC (17:0), and CER (17:0) at 1 µM as internal standards. The homogenate was shaken and incubated at 37 °C for 20 minutes followed by centrifugation at 15,000×g for another 15 minutes. The lower organic phase was collected and evaporated to dryness under vacuum. The residue was then suspended with 100 µL chloroform:methanol (1:1, vol/vol) solution and then diluted with isopropanol:acetonitrile: H ₂ O (2:1:1, vol/vol/vol) solution before injection. Lipidomics analysis was performed on an Acquity UPLC/Synapt G2 Si HDMS QTOFMS system (Waters Corp., Milford, MA) equipped with electrospray ionization (ESI) source. Separation was achieved on an Acquity UPLC CSH C18 column (100×2.1-mm internal diameter, 1.7 mm; Waters Corp.). The mobile phase was a mixture of acetonitrile/water (60/40, vol/vol; A) and isopropanol/acetonitrile (90/10, vol/vol; B), and both A and B contained 10 mM ammonium acetate and 0.1% formic acid. The gradient elution program consisted of a 2-minute linear gradient of 60% A to 57% A to 50% A at 2.1 minutes, a linear decrease to 46% A at 12 minutes to 30% A at 12.1 minutes, and a linear decrease to 1% A at 18 minutes before returning to initial conditions at 18.5 minutes to equilibrate the column. The column temperature was maintained at 55 °C, and the flow rate was 0.4 mL/min. Mass spectrometry data was acquired in both the positive and negative ESI modes at a range of m/z 100-1200.

2) For Kidney Bile Acid Composition:

Kidney tissue was homogenized with 200 µL acetonitrile containing 1 µM

trichloroacetic acid-d5. The samples were centrifuged at 15,000×g for 15 minutes at 4 °C; 40 µL supernatant was collected and diluted ten times with 0.1% formic acid before analysis. Concentrations of bile acids were determined by an Acquity UPLC/Xevo G2 QTOFMS system (Waters Corp.) with an ESI source. An Acquity BEH C18 colum (100×2.1-mm internal diameter, 1.7 mm; Waters Corp.) was applied for chromatographic separation. A mixture of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) was used as the mobile phase. The gradient elution was started from 80% A for 4 minutes, decreased linearly to 60% A over 11 minutes, to 40% A over the next 5 minutes, and to 10% A for the succeeding 1 minute, and finally, increased to 80% A for 4 minutes to re-equilibrate the column. Column temperature was maintained at 45 °C, and the flow rate was 0.4 mL/min. Mass spectrometry detection was operated in negative mode. A mass range of m/z 50-1000 was acquired.

^ NF-κB Transcriptional Activity Assay

Nuclear protein extracts were prepared from kidney tissue. The nuclear extracts were used for the measurement of NF-κB transcriptional activity with a kit from Marligen

Biosciences (Rockville, MD) according to the manufacturer’s instructions.

^ Oxidized Protein Analyses

The amount of oxidized proteins in kidney homogenates was determined by using an OxyElisa Oxidized Protein Quantitation Kit (Millipore) according to the manufacturer’s instructions.

^ Quantification of Morphology

All quantifications were performed in a masked manner. Using coronal sections of the kidney, 30 consecutive glomeruli per mouse with six mice per group were examined for evaluation of glomerular mesangial expansion. The index of the mesangial expansion was defined as the ratio of mesangial area to glomerular tuft area. The mesangial area was determined by assessment of the periodic acid-Schiff-positive and nucleus-free area in the mesangium using ScanScope image analyzer (Aperio Technologies, Vista, CA).

^ Autofluorescence FLIM, SHG, and Third Harmonic Generation Measurements Using the Deep Imaging via Enhanced-Photon Recovery Microscope

Autofluorescence fluorescence lifetime imaging microscopy (FLIM), second harmonic generation (SHG), and third harmonic generation (THG) signals were acquired using the Deep Imaging via Enhanced-Photon Recovery microscope at the Laboratory of Fluorescence Dynamics, University of California, Irvine. This microscope is especially useful for harmonic imaging, both second and third harmonic, because of its transmission geometry. Briefly, the system is on the basis of a custom-made upright laser-scanning microscope equipped with the special Deep Imaging via Enhanced-Photon Recovery detector. A short pulsed two-photon laser (Insight Deep See; Spectra-Physics) is used as the excitation source, and an Acousto- Optic Modulator (AA Opto-Electronic) is used to modulate excitation power. The samples were excited with a 40X, 0.8 NA water objective (Olympus) for harmonic and FLIM measurements. The sample is placed directly on top of the detector assembly input window below the objective, and two photon-induced fluorescence, SHG, and THG are detected by a large area photomultiplier (PMT; R7600P-300; Hammatsu). The detector assembly consists of a sealed chamber with the filter wheel/shutter inside and the housing with PMT. The refractive index matching liquid fills the inside of the housing, and removes loss of photons due to internal reflections and thus, achieves efficient collection of photons. Two BG39 filters serve as input and output windows of the chamber and block NIR excitation light from entering PMT and transmitting UV and visible fluorescence as well harmonic signals. The only optical elements in the detector assembly are BG39 filters and the glss filter of the filter wheel, allowing detection of emitted photons from 320- to 650-nm-wavelenth range. The transmission geometry of detection system allows more efficient detection of SHG and THG signals due to forward propagating nature of the harmonic signals.

The signal from the PMT is collected using an FLIMBox and directly transferred to the phasor plot. Briefly, this method of lifetime analysis involves transferring the

fluorescence intensity decays in the Fourier space and plotting the Fourier components against each other. This results in a phasor plot as shown in Figure 14B, where each point results from a pixel of the image. The SHG and THG signals are coherent, and the signal resembles that of the laser pulse, which is measured as zero lifetime; hence, the phasor points originating from SHG appear at s = 0, g = 1 in the phasor plot. However, the autofluorescence from the tissue has a nonzero lifetime and appears inside the semicircle. A distribution in the phasor plot can then be selected using either a cursor or a continuous distribution cursor, and the images can be colored accordingly. Phasor approach toward FLIM is a fit-free approach, and increases the speed of the analysis and decreases the computational difficulty associated with FLIM technique. Free NADH and bound NADH have lifetimes of 0.4 and approximately 3.4 ns, respectively, and their individual fluorescence intensity decay seems close to the universal circle. In Figure 14B, the phasor positions of free and bound NADH are represented by the blue and the red cursors, respectively and the line joining the two cursors is called the metabolic trajectory. The distribution of the points along the metabolic trajectory can be then transformed to a distribution depicting the number of pixels belonging to a certain fraction of free NADH using the law of linear addition in phasor space. The distribution of free NADH can then be compared with the difference in metabolism of different samples.

The state of fibrosis was analyzed by a ratiometric method. In this new method, a ratio of the area covered by SHG to the area covered by FLIM is calculated. This analysis takes into account of both collagen accumulation due to fibrosis and the changes in tissue architecture. The higher the value of this ratio, the higher the fibrosis. The green and red colors in Figure 14B were used to show the SHG and FLIM signals, respectively, and thus, the ratio has been defined as f _green /f _red . The data collection and analysis were carried out by using SimFCS developed by the Laboratory for Fluorescence Dynamics, University of California, Irvine.

The differences in spatial accumulation of all of the lipids and the long lifetime species, which are representative of ROS and lipid oxidation, are imaged through the THG and FLIM, respectively.

SHG imaging was carried out with a 710-nm pulsed laser for excitation and a combination of UG-11 and BG39 filters for the efficient detection of 355-nm SHG photons. The combination of BG39 and UG-11 creates a spectral window of observation with maxima around 355 nm. Thus, the SHG signal (green) when excited at 710 nm can be very efficiently collected using these two filters. THG was achieved by exciting the samples at 1050 nm and detecting the THG signal at 1050/3 = 350 nm using the same filter settings as those of SHG. Two-photon autofluorescence FLIM was achieved by using the same 710-nm excitation as that of the SHG and the BG39 filter of the detection assembly (λ _EM = 300-650 nm). Each of the images was taken with a 360-µm field of view, 32-µs pixel dwell time, and 20 repeat scans.

^ Statistical analysis

Results were presented as the means ± SE for at least three independent experiments. Data were analyzed by ANOVA and Student-Newman-Keuls tests for multiple comparisons or by Student's t test for unpaired data between two groups. Statistical significance was accepted at the P < 0.05 level.

The experiments of the present disclosure were performed according to methods of the art, for example as in, Wang XX. et al., J. Biol. Chem.292, 12018 (2017) or Wang XX. et al., J. Am. Soc. Nephrol.29, 118 (2018)– the entire contents of each of which are incorporated herein by reference in their entirety. Example 3. Caloric restriction prevents age-related decreases in renal FXR and TGR5 expression.

Compared to 5-month-old ad lib fed mice, 24-month-old ad lib fed mice had significant decreases in FXR and TGR5 mRNA expression (see, e.g., Wang (2017)). These decreases were prevented in 24-month-old mice with life-long caloric restriction (Figures 1A and 1B). The effects of aging and caloric restriction on FXR were also illustrated by western blotting at the protein level (Figures 1C and 1D). Example 4. A compound of the present application reverses age-related increase in urinary albumin excretion.

22-month-old ad lib fed mice were treated with a compound of the present application (e.g., Compound 1-Na) for 2 months (see, e.g., Wang (2017)). The treatment induced a significant decrease in urinary albumin, similar to beneficial effects achieved with life-long caloric restriction (Figure 2A). The compound of the present application (e.g., Compound 1- Na) also prevented the age-related decrease in synaptopodin expression, as determined by immunofluorescence microscopy (Figure 2B). The beneficial effects were associated with prevention of the age-related increases in the level of transforming growth factor-β (TGF-β), fibroblast specific protein-1 (FSP-1), and fibronectin (Figures 2C and 2D). Example 5. A compound of the present application reverses age-related decrease in mitochondrial biogenesis and function.

Mitochondrial to nuclear DNA ratio, a hallmark of mitochondrial biogenesis, and the level of nuclear respiratory factor 1 (NRF1), the master regulator of mitochondrial biogenesis, were significantly decreased in the kidneys of 24-month-old ad lib fed mice (Figures 3A and 3B) (see, e.g., Wang (2017)). These decreases were reversed by treatment with a compound of the present application (e.g., Compound 1-Na) (Figures 3A and 3B). The treatment concomitantly increased the activated AMPK level (Figures 3C and 3D), and prevented or reversed the age-related decreases in SIRT1 mRNA (Figure 3E) and the nuclear hormone receptor estrogen related receptor-alpha (ERR-α) mRNA (Figure 3F). Furthermore, treatment of 22-month-old ad lib fed mice with a compound of the present application (e.g., Compound 1-Na) for 2 months prevented or reversed the age-related decrease in PGC-1α expression (Figures 3G, 3J, and 3K), and mitochondrial SIRT3 expression (Figures 3G, 3H, and 3I). The increase in SIRT3 was accompanied by increases in medium-chain Acyl-CoA dehydrogenase (MCAD) protein, an important mediator of mitochondrial fatty acid β-oxidation (Figure 3L). At the same time the compound of the present application (e.g., Compound 1-Na) also prevented/reversed the age-related increase in acetylated form of mitochondrial isocitrate dehydrogenase (acetyl-IDH2/IDH2), another target of SIRT3 activity (Figures 3M and 3N). This is associated with the reversal of the decreased mitochondrial complex I and complex IV activity in aged kidneys by the 2-month treatment, at levels identical to life-long caloric restriction (Figures 3O and 3P). Example 6. A compound of the present application decreases inflammation in human podocytes conditioned with aging serum.

Human podocytes were treated with serum from young (4-month old) or old (28- month old) mice (see, e.g., Wang (2017)). Aging serum increased the expression of TNF-α, TLR2, and TLR4 (Figure 4). Treatment with a compound of the present application (e.g., Compound 1-Na) prevented these changes (Figure 4), indicating a direct effect of FXR and TGR5 activation on the podocytes in culture. Example 7. FXR and TGR5 expression are increased in long-lived Ames dwarf mice.

The Ames dwarf mice that exhibit delayed aging and extended longevity were studied to determine if FXR and TGR5 expression were regulated similar to caloric restriction (see, e.g., Wang (2017)). Similar to caloric restriction, the FXR and TGR5 mRNA levels increased in the kidneys of Ames dwarf mice (Figures 5A and 5B). In addition, the genes involved in the mitochondrial biogenesis and function, including NRF1, SIRT1, PGC1α, ERRα, SIRT3, COX4, and LCAD, also increased in the Ames kidneys, consistent with the findings with the treatment of aging C57BL/6 mice with a compound of the present application (e.g.,

Compound 1-Na) (Figures 5C-5I). Example 8. FXR mRNA and protein expression is decreased in human nephropathy in diabetes and obesity.

FXR mRNA is markedly reduced in both glomeruli and tubules in kidney biopsies obtained from human subjects with nephropathy associated with diabetes and obesity, after laser capture microdissection, RNA extraction, and quantitative RT-PCR (Figure 6A) (see, e.g., Wang (2018)). Immunohistochemistry for FXR in kidney biopsies obtained from human subjects with diabetic nephropathy was also performed. FXR staining in control subjects was concordant with the expression reported from rat tubular segments with predominant expression in the S1 segment of the proximal tubule and cortical thick ascending limb of the loop of Henle, with less expression in the S2 and S3 segment and distal convoluted tubule. A significant decrease in FXR protein expression in the cortical tubular epithelium across segments, including in the proximal tubular epithelium, was found in subjects with diabetic nephropathy (Figure 6B and Figure 6C). Further, changes in TGR5 protein expression were not observed in spite of a decrease in TGR5 mRNA (Figure 6D). Example 9. Distinct pathways linked to FXR or TGR5 activation in diabetic kidneys.

To determine if FXR versus TGR5 agonists activate differential pathways in the diabetic kidney, DBA/2J mice were treated with STZ-induced hyperglycemia with Compound 2 (obeticholic acid), Compound 3, or Compound 1-Na (6α-ethyl-3α, 7α, 23-trihydroxy-24- nor-5-β-cholan-23 sulfate sodium salt) (see, e.g., Wang (2018)). RNA-Seq analysis indicated that Compound 2 (Figure 7B) and Compound 3 (Figure 7C) activate distinct pathways that are relevant for the pathogenesis and treatment of diabetic kidney disease. Compound 1-Na can further activate an additional set of pathways (Figure 7A and 7D). In the protein level, both Compound 2 and Compound 1-Na, but not Compound 3, can regulate lipogenesis pathway mediated by SREBP-1 and targets SCD-1, SCD-2, and FIT-1 mRNA (Figure 7E, Figure 7F, Figure 7G, Figure 7H, Figure 7I). However, Compound 3 and Compound 1-Na, but not Compound 2, can both induce mitochondrial biogenesis pathway as shown by increases in SIRT1, PGC-1α, and ERR-α protein expression (Figure 7J, Figure 7K, Figure 7L, Figure 7M, Figure 7N, and Figure 7O). Thus, Compound 1-Na can simultaneously activate both FXR and TGR5 signaling and their nonoverlapping pathways, with potential additive effects (Figure 7P). Example 10. Compound 1-Na decreases albuminuria and prevents renal histopathologic alterations and renal fibrosis in diabetic DBA/2J mice.

DBA/2J mice with STZ-induced hyperglycemia fed a standard chow diet (10 kcal% fat, complex carbohydrates) develop significant proteinuria, but only develop very minimal renal histopathologic changes (see, e.g., Wang (2018)). When these mice were fed a Western diet (42 kcal% milkfat, 34% sucrose, 0.20% cholesterol; approximating the human Western diet), they developed marked albuminuria, which was normalized by Compound 1-Na treatment (Figure 8A). Treatment with Compound 1-Na also decreased the glomerular area and mesangial matrix expansion (Figure 8B, Figure 8C, and Figure 8D). Masson trichrome staining showed patchy fibrosis in the tubular interstitium, which was nearly absent in the kidneys of Compound 1-Na treated mice (Figure 8E). Label-free imaging of kidney sections with two-photon excitation and second harmonic generation (SHG) microscopy also showed accumulation of extracellular matrix proteins in the tubular interstitium, which was prevented by treatment with Compound 1-Na (Figure 8F).

Immunofluorescence microscopy showed increased fibronectin and type 4 collagen in the glomeruli of the diabetic kidney which were prevented by Compound 1-Na treatment (Figure 8G, Figure 8H, Figure 8I, and Figure 8J). In addition, immunohistochemistry showed increased staining with α-smooth muscle actin (α-SMA), which was prevented by Compound 1-Na as well (Figure 8K).

In diabetic DBA/2J mice, staining with WT-1, a nuclear podocyte marker, showed a significantly reduced podocyte density in diabetic mice, which was rescued by Compound 1- Na treatment (Figure 8L and Figure 8M). Podocyte loss was also confirmed by

immunofluorescence staining of the podocyte marker nephrin in kidney sections, showing reduced expression in diabetic kidneys, which was prevented by Compound 1-Na treatment (Figure 8N). Example 11. Compound 1-Na prevents activation of profibrotic signaling pathways in diabetic DBA/2J mice.

In diabetic DBA/2J mice, Compound 1-Na treatment blocked the increase of TGF-β expression in diabetic kidneys as well as its target gene CTGF (Table 1) (see, e.g., Wang (2018)). Expression of two myofibroblast markers, α-SMA (Figure 8K) and FSP-1 (Table 1), was increased in diabetic DBA/2J mice and decreased after Compound 1-Na treatment. The expression of their transcriptional regulators Snail and Zeb1 was also downregulated by Compound 1-Na treatment (Table 1). Table 1. Metabolic Parameters and Renal Gene Expression in DBA/2J Mice

Example 12. Compound 1-Na modulates renal lipid metabolism and prevents renal triglyceride and cholesterol accumulation in diabetic DBA/2J mice.

Diabetic DBA/2J mice were shown to have increased kidney neutral lipid accumulation in both glomeruli and tubulointerstitium by oil red O staining (Figure 9A) (see, e.g., Wang (2018)). The lipid accumulation was mediated by increases in SCD-2 as well as ChREBP-β and liver pyruvate kinase (Table 1). Biochemical analysis of kidney lipid extracts showed increased kidney triglyceride and cholesterol accumulation, which was significantly decreased by Compound 1-Na treatment (Figure 9B and Figure 9C). The effects of

Compound 1-Na in decreasing renal triglyceride and cholesterol content were mediated by coordinated effects inducing (i) decreased expression of SREBP-1c and its target genes SCD- 1 and SCD-2, which mediate fatty acid and triglyceride synthesis (Table 1); (ii) decreased expression of liver pyruvate kinase, which also mediates fatty acid and triglyceride synthesis (Table 1); (iii) decreased expression of SREBP-2, which mediates cholesterol synthesis (Table 1); (iv) increased expression of lipolysis gene LPL (Table 1); and (v) decreased expression of lipid droplet formation gene FIT-1 (Table 1).

Compound 1-Na also decreases serum triglycerides and LDL cholesterol in diabetic DBA/2J mice. STX treatment of DBA/2J mice fed a Western diet resulted in marked increases in serum glucose, triglyceride, and cholesterol levels, with most of the cholesterol derived from LDL (14.0 ± 2.1 mg/dl in control versus 654 ± 79 mg/dl in diabetic mice) (Table 1). Treatment with Compound 1-Na did not decrease serum glucose levels in diabetic DBA/2J mice but significantly decreased plasma triglyceride, total cholesterol, and LDL cholesterol levels (654 ± 79 mg/dl in diabetic mice versus 40.5 ± 2.5 mg/dl in diabetic mice treated with Compound 1-Na) (Table 1). Example 13. Compound 1-Na prevents inflammation, oxidative stress, and endoplasmic reticulum stress in diabetic DBA/2J mice.

Compound 1-Na markedly decreased the expression of macrophage marker CD68 in diabetic kidneys (Figure 10A) (see, e.g., Wang (2018)). This was consistent with the inhibition by Compound 1-Na treatment of NF-κB p65 and p50 heterodimeric complexes expression (Figure 10B and Figure 10C) and NF-κB activity, the master transcription factor regulating inflammation (Figure 10D). The expression of NF-κB-dependent proinflammatory mediators, like intercellular adhesion molecule-1 and cyclooxygenase-2, was also

significantly decreased by Compound 1-Na treatment (Table 1). In addition, Compound 1-Na modulates oxidative stress, as shown by reduced total protein carbonylation in diabetic kidneys from treated mice (Figure 10E) and decreased NADPH oxidase Nox-2 and p22-phox mRNA expression (Figure 10F and Figure 10G). However, NADPH oxidase Nox-4 was not changed (Figure 10H). Endoplasmic reticulum (ER) stress is increased in the kidneys of diabetic mice as determined by increased expression of phospho-EIF-2α-to-total EIF-2α protein ratio (Figure 11E and Figure 11F) and CHOP mRNA level (Table 1). Treatment with Compound 1-Na decreased phospho-EIF-2α-to-total EIF-2α protein ratio (Figure 11E and Figure 11F), which was also associated with increased glucose regulated/binding Ig protein- 78 (BiP) and spliced form of X-box binding protein-1 mRNA levels (Table 1). Example 14. Compound 1-Na modulates hypoxia signaling in diabetic DBA/2J mice.

The mRNA abundance of HIF-1α and HIF-2α was significantly increased in diabetic DBA/2J mice (see, e.g., Wang (2018)). Treatment with Compound 1-Na prevented the increased expression of HIF-1α and HIF-2α in diabetic kidneys (Figure 11A and Figure 11B). As a downstream target for HIF signaling, Glut1 expression was also increased in diabetic kidneys but reversed by Compound 1-Na treatment (Figure 11C and Figure 11D). Example 15. Renal effects of Compound 1-Na in the db/db mouse model of type 2 diabetes mellitus and obesity.

The therapeutic efficacy and renal effects of Compound 1-Na were determined in a well-established model of type 2 diabetes mellitus associated with obesity (see, e.g., Wang (2018)). Compound 1-Na stimulated FXR and TGR5 mRNA in both db/m and db/db mice (Figure 12A and Figure 12B). Interestingly, although FXR mRNA was increased in db/db mice, FXR protein abundance as determined by FXR immunohistochemistry was decreased in db/db mice (- 35.2% relative to db/m), and Compound 1-Na treatment activated FXR protein expression to levels seen in nondiabetic db/m mice (Figure 12C). Compound 1-Na treatment of db/db mice did not alter blood glucose levels, but significantly decreased plasma total cholesterol and triglyceride levels (Table 2). In addition, treatment of db/db mice with Compound 1-Na resulted in significantly decreased albuminuria (Figure 12D). Treatment with Compound 1-Na also decreased mesangial matrix expansion (Figure 12E and Figure 12F), podocyte loss as shown by synaptopodin immunofluorescence microscopy (Figure 12G), renal fibrosis indicated by the decreased collagen 1 (Figure 12I and Figure 12J), and collagen 3 (Figure 12K and Figure 12L) protein abundance as determined by

immunohistochemistry and extracellular matrix protein fibronectin, but not collagen 4, expression as determined by immunofluorescence microscopy (Figure 12M, Figure 12N, Figure 12O and Figure 12P). Treatment with Compound 1-Na decreased oxidative stress as shown by significant changes in urinary H2O2 and TBARS level (Figure 12Q and Figure 12R). In addition, treatment with Compound 1-Na significantly increased CD163 and CD206 expression in macrophages (Table 2) suggesting that FXR/TGR5 activation enhances anti- inflammatory M2 macrophages in the db/db mice. Table 2. Metabolic Parameters and Renal Gene Expression in db/db Mice

Example 16. Compound 1-Na induces mitochondrial biogenesis and metabolism pathways in db/db mouse mode of type 2 diabetes mellitus and obesity.

Treatment of db/db mice with Compound 1-Na increased phospho-AMPK-to-AMPK protein ratio (Figure 13A and Figure 13B), nicotinamide phosphoribosyl transferase, the rate limiting enzyme in NAD ⁺ biosynthesis (Table 2), SIRT1 mRNA (Table 2) and protein (Figure 13C and Figure 13D), and SIRT3 mRNA (Table 2) (see, e.g., Wang (2018)). Compound 1-Na treatment also increased PGC-1α mRNA (table 2) and protein (Figure 13E and Figure 13F), ERR-α, and Nrf1 mRNA (Table 2), the transcriptional regulators of mitochondrial biogenesis and activity, as well as several enzymes that mediated fatty acid and glucose oxidation, including carnitine palmitoyltransferase-1A, pyruvate dehydrogenase kinase 4, long-chain acyl CoA dehydrogenase, and acetyl CoA synthetase 2 (Table 2). Example 17. Compound 1-Na prevents mitochondrial dysfunction, oxidative stress, inflammation, and fibrosis in mice with diet-induced obesity.

In mice fed a high-fat diet, there was an increase in free, nonmitochondria-bound NADH as determined by label-free imaging with fluorescence lifetime imaging microscopy (FLIM) (Figure 14A and Figure 14B) (see, e.g., Wang (2018)). The increase in free NADH is indicative of mitochondrial dysfunction and oxidative stress. There was also increased expression of the proinflammatory TLR4 (Figure 14C). These alterations were associated with increased renal fibrosis as determined by label-free imaging with SHG-FLIM (Figure 14D and Figure 14E). Treatment with Compound 1-Na prevented the increase in NADH free fraction (Figure 14A), TLR4 expression (Figure 14C), and fibrosis (Figure 14D and Figure 14E). Example 18. Compound 1-Na prevents ceramide and triglyceride accumulation and alters ceramide and triglyceride composition in mice with diet-induced obesity.

In mice fed a high-fat diet, there was a significant increase in C16:0 ceramide level (Figure 14F) (see, e.g., Wang (2018)). Treatment with Compound 1-Na prevented the increases in total and individual ceramide species levels (Figure 14F). In mice fed a high-fat diet, there were also significant increases in total and individual triglyceride species levels (Figure 14G and Figure 14H). Treatment with Compound 1-Na prevented the increases of most triglyceride species but most significantly, the C52:1 and C54:2 triglyceride species levels (Figure 14G and Figure 14H). Example 19. Compound 1-Na prevents bile acid accumulation and alters bile acid composition in mice with diet induced obesity.

In mice fed a high fat diet, there was a significant increase in total bile acid levels (Figure 15A) (see, e.g., Wang (2018)). The increase in absolute and relative trichloroacetic acid levels was most marked and significant (Figure 15A, Figure 15B, Figure 15C and Figure 15D). Treatment with Compound 1-Na induced significant decreases in total bile acid levels and absolute and relative trichloroacetic acid, T-α-MCA, T-β-MCA, T-DCA, and T-HDCA levels (Figure 15A, Figure 15B, Figure 15C and Figure 15D). To explore for potential mechanisms of these changes in total and individual bile acid composition, the expression was measured of the renal bile acid synthesis and bile acid transporter genes. High-fat diet induced decreases in Cyp7B1 mRNA, which mediates bile acid synthesis, and ASBT mRNA, which mediates bile acid transport from the urine (Figure 15E, Figure 15F, Figure 15G, Figure 15H, Figure 15I, Figure 15J and Figure 15K). Treatment with Compound 1-Na did not cause any significant changes in Cyp7B1, ASBT mRNA, or other bile acid synthesis and bile acid transporter genes (Figure 15E, Figure 15F, Figure 15G, Figure 15H, Figure 15I, Figure 15J and Figure 15K).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.