The farnesoid X receptor modulates renal lipid metabolism and diet-induced renal inflammation, fibrosis, and proteinuria

Abstract

Diet-induced obesity is associated with proteinuria and glomerular disease in humans and rodents. We have shown that in mice fed a high-fat diet, increased renal expression of the transcriptional factor sterol-regulatory element binding protein-1 (SREBP-1) plays a critical role in renal lipid accumulation and increases the activity of proinflammatory cytokines and profibrotic growth factors. In the current study, we have determined a key role of the farnesoid X receptor (FXR) in modulating renal SREBP-1 activity, glomerular lesions, and proteinuria. We found that feeding a Western-style diet to DBA/2J mice results in proteinuria, podocyte loss, mesangial expansion, renal lipid accumulation, and increased expression of proinflammatory factors, oxidative stress, and profibrotic growth factors. Treatment of these mice with the highly selective and potent FXR-activating ligand 6-α-ethyl-chenodeoxycholic acid (INT-747) ameliorates triglyceride accumulation by modulating fatty acid synthesis and oxidation, improves proteinuria, prevents podocyte loss, mesangial expansion, accumulation of extracellular matrix proteins, and increased expression of profibrotic growth factors and fibrosis markers, and modulates inflammation and oxidative stress. Our results therefore indicate that FXR activation could represent an effective therapy for treatment of abnormal renal lipid metabolism with associated inflammation, oxidative stress, and kidney pathology in patients affected by obesity.

due to the consumption of a Western-style diet and sedentary lifestyles, obesity is rapidly becoming the most important health problem challenging developed countries. Although obesity is often associated with diabetes and hypertension, the two most common risk factors for the development of end-stage renal disease, obesity itself has been suggested as an independent risk factor for developing chronic kidney disease (35, 47, 48). Early in the course of obesity, structural and functional changes similar to diabetic kidney disease occur (17). These changes, considered precursors to more severe renal injury, include glomerular hyperfiltration, glomerular basement membrane thickening, mesangial cell proliferation, mesangial matrix thickening, and expansion of Bowman's capsule. Massive obesity has been associated with the eventual development of focal and segmental glomerulosclerosis, even in the absence of diabetes (24, 34).

Although incompletely understood, several hormonal and metabolic factors have been shown to contribute to the pathogenesis of obesity-related renal disease, including oxidative stress, angiotensin II, inflammatory cytokines, hyperinsulinemia/insulin resistance, hyperglycemia/diabetes, and hyperlipidemia (2, 4, 35, 52). In addition, an important pathogenic role for renal lipid accumulation has also been proposed in our earlier studies (20–23, 36, 44, 49). We demonstrated that increased renal expression of sterol-regulatory element-binding proteins (SREBP-1 and SREBP-2) is the key factor linking increased fatty acid and cholesterol synthesis and accumulation of lipids to development of nephropathy. Genetic manipulation of SREBP, using SREBP-1c null mice, largely prevented renal accumulation of lipids and expression of proinflammatory cytokines and profibrotic growth factors. These studies therefore imply that alterations in renal lipid metabolism mediated by SREBP-1 play an important role in the pathogenesis and progression of renal disease in type 1 diabetes mellitus, obesity and insulin resistance, type 2 diabetes mellitus, and aging.

The farnesoid X receptor (FXR) is a bile acid-activated nuclear receptor which plays an important role in regulating bile acid metabolism (14, 29). FXR has been shown to control lipid metabolism by a mechanism involving repression of hepatic SREBP-1c expression (51, 54). FXR is also involved in the regulation of carbohydrate metabolism, including inhibition of gluconeogenesis (11, 28, 42, 55). Moreover, FXR activation prevents liver fibrosis (12) and atherosclerotic lesions (16, 26).

FXR is a potential pharmacological target for the treatment of obesity and metabolic syndrome. The kidney has a high expression level of FXR. FXR and SREBPs coexist in the glomeruli and proximal tubule cells and are expressed in cultured mouse mesangial cells and podocytes (22). The purpose of this study was therefore to determine the role of FXR activation in a mouse model of nephropathy associated with diet-induced obesity (DIO). Like human obesity, DIO is a polygenic condition reflecting the interaction of genetic predisposition with the environment (39), making it a valuable model in developing new therapeutic strategies. Here, we demonstrate that FXR activation ameliorates obesity-related renal disease, regulating renal lipid metabolism, fibrosis, inflammation, and oxidative stress.

MATERIALS AND METHODS

Animals and treatments.

Male DBA/2J mice were obtained from Jackson Laboratories (Bar Harbor, ME). They were maintained on a 12:12-h light-dark cycle and fed a control low-fat diet (CON; TD76329) or a high-fat, high-cholesterol diet (WD; TD88137) obtained from Harlan-Teklad (Madison, WI) for 12 wk with the treatments of 1) vehicle only and 2) synthetic FXR ligand 6-α-ethyl-chenodeoxycholic acid (INT-747; Intercept Pharmaceuticals, New York, NY) (33): 10 mg/kg body wt administered via gavage once a day.

Homozygous male FXR null mice (FXR^−/−) of 20 wk of age and sex- and age-matched wild-type mice bred on the C57BL/6 genetic background (41) were maintained on a 12:12-h light-dark cycle and fed a WD diet for 1 wk with the treatment of either vehicle only or INT-747 at 10 mg/kg body wt administered via gavage once a day.

The animal studies and the protocols were approved by the Institutional Review Boards at the University of Colorado Denver.

Blood and urine chemistries.

Blood glucose levels were measured by means of a Glucometer Elite XL (Bayer, Tarrytown, NY). Plasma triglyceride and cholesterol [total cholesterol (TC), HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C)] were measured with kits from Wako Chemical (Richmond, VA). Urine albumin and creatinine concentrations were determined using kits from Exocell (Philadelphia, PA). Results are expressed as the urine albumin-to-creatinine ratio (μg/mg).

RNA extraction and quantitative real-time PCR.

Total RNA was isolated from the kidneys using the SV total RNA isolation system from Promega (Madison, WI), and cDNA was synthesized using reverse transcript reagents from Bio-Rad Laboratories (Hercules, CA). The mRNA level was quantified using a Bio-Rad iCyCler real-time PCR machine. Cyclophilin was used as an internal control, and the amount of RNA was calculated by the comparative threshold cycle (C_T) method as recommended by the manufacturer. All the data were calculated from triplicate reactions. Primer sequences used have been described previously (22) or are available from the authors upon request.

Protein electrophoresis and Western blotting.

Equal amounts of protein samples were subjected to SDS-PAGE, and they were then transferred to nitrocellulose membranes. After blocking with 5% fat-free milk powder in 0.1% Tween 20 in Tris-buffered saline (20 mmol/l Tris-Cl and 150 mmol/l NaCl, pH 7.4), the blots were incubated with antibodies against podocin (1:200; G20; Santa Cruz Biotechnology, Santa Cruz, CA), transforming growth factor-β (TGF-β) type II receptor (TGFβR2; 1:200; SC-400, Santa Cruz Biotechnology), α-smooth muscle actin (α-SMA; 1:1,000; Sigma, St. Louis, MO), or manganese superoxide dismutase (MnSOD; 1:200; N20; Santa Cruz Biotechnology). Corresponding secondary antibody was visualized using enhanced chemiluminescence (Pierce, Bradford, IL). The signals were quantified with a PhosphorImager with a chemiluminescence detector and the accompanying densitometry software (Bio-Rad).

Lipid extraction and measurement of lipid composition.

Lipids from the kidneys were extracted by the method of Bligh and Dyer, as we have previously described (20–23, 36). Triglyceride and cholesterol content was measured using kits from Wako Chemical.

Periodic acid-Schiff staining, immunohistochemistry, and immunofluorescence microscopy.

Sections (4-μm thick) cut from 10% formalin-fixed, paraffin-embedded kidney samples were used for periodic acid-Schiff (PAS) staining and podocyte nuclei detection with a rabbit polyclonal anti-Wilm's tumor 1 (WT1) antibody (Santa Cruz Biotechnology) using an ABC Staining Kit (Vector Laboratory, Dedham, MA). Frozen sections were used for immunostaining for podocin (Sigma), synaptopodin (Sigma), fibronectin (Sigma), collagen IV (Rockland Immunochemicals, Gilbertsville, PA), and platelet-derived growth factor receptor-β chain (PDGFRβ; eBioscience, San Diego, CA) and imaged with a laser-scanning confocal microscope (LSM 510, Zeiss, Jena, Germany).

Quantification of morphology.

All quantifications were performed in a blinded manner. Using coronal sections of the kidney, 30 consecutive glomeruli/mouse (6 mice/group) were examined for evaluation of glomerular mesangial expansion. The index of the mesangial matrix was defined as the ratio of mesangial to glomerular tuft area. The mesangial area was determined by assessment of the PAS-positive and nucleus-free area in the mesangium using a ScanScope image analyzer (Aperio Technologies, Vista, CA).

Statistical analysis.

Results are presented as 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.

RESULTS

INT-747 treatment of WD-fed DBA mice reduces body weight gain and modulates serum lipid chemistry.

To investigate how FXR activation impacts WD-fed DBA mice, we performed a 12-wk treatment with the selective FXR agonist INT-747 in 8-wk-old DBA/2J mice fed a WD. The growth curve shows that WD-fed mice had significant weight gain compared with mice fed the control diet. INT-747 treatment provided a degree of protection from weight gain, decreasing it significantly (Fig. 1, Table 1). WD mildly increased plasma triglyceride levels in DBA mice but caused a significant increase in plasma TC, blood glucose, and insulin levels. INT-747 significantly reduced plasma cholesterol and showed a trend toward reduction in plasma triglyceride and blood glucose levels (Table 1). A significant reduction in HDL-C and LDL-C levels was also induced by INT-747 treatment (Table 1). No change was observed in the HDL-C/TC ratio. However, the LDL-C/TC ratio was increased in the WD-fed group and decreased with INT-747 treatment.

Fig. 1.

Changes in body weight in DBA/2J mice placed on a low-fat diet (control diet; CON) or a high-fat, high-cholesterol diet (Western-style diet; WD) for 12 wk. ♦, CON; ■, WD; ▴, WD plus INT-747. *P < 0.05 vs. CON (n = 6–8 mice/group).

Table 1.

Metabolic parameters

	DBA Control	DBA WD	DBA WD+INT-747
Body weight, g	27.9±0.68	42.2±0.94*	38.3±0.85†
Kidney weight, g	0.53±0.03	0.69±0.04*	0.64±0.02
Kidney/body weight ratio, %	1.84±0.066	1.62±0.085	1.68±0.078
Blood glucose, mg/dl	83.4±5.00	118.1±6.86*	109.6±5.92
Plasma insulin, ng/ml	2.57±0.64	10.04±0.91*	9.89±1.59
Plasma TG, mg/dl	151.6±18.5	192.1±26.4	185.4±23.5
Plasma TC, mg/dl	100.1±3.45	178.0±11.2*	145.1±4.34†
Plasma HDL-C, mg/dl	71.8±4.55	113.7±4.18*	99.4±4.50†
Plasma HDL-C/TC, %	71.8	63.9	67.7
Plasma LDL-C, mg/dl	21.5±1.67	55.3±4.38*	38.0±2.58†
Plasma LDL-C/TC, %	21.5	31.1*	25.9†

Values are means ± SE (n = 6 mice/group). WD, Western-style diet; TG, total glucose; TC, total cholesterol; HDL-C, HDL-cholesterol; LDL-C, LDL-cholesterol.

^*P < 0.05 vs. control.

^†P < 0.05 vs. WD.

Treatment of WD-fed DBA mice with INT-747 improves proteinuria and ameliorates renal structural changes.

DBA/2J mice with DIO developed moderate albuminuria after 12-wk WD, which was significantly decreased and nearly normalized by INT-747 treatment (Fig. 2A). An important mechanism causing albuminuria is podocyte dysfunction, prompting us to examine podocyte marker expression in DBA/2J kidney lysates. As shown in Fig. 2B, podocin protein expression was reduced in the WD-fed group but significantly increased by INT-747 treatment. This was confirmed by immunostaining of glomeruli with antibodies to podocin and synaptopodin (Fig. 2, C and D). These results are consistent with the improvement in albuminuria induced by INT-747 treatment, as podocin is a key component of the slit diaphragm and synaptopodin is a regulator of actin dynamics for podocyte foot processes, both of critical importance for sustained function of the glomerular filtration barrier to proteinuria (19, 27, 31). Podocyte loss was also confirmed by staining with WT1, a nuclear podocyte marker, showing a significantly reduced podocyte density in the WD-fed group which was prevented by INT-747 treatment (Fig. 2E). Glomerular mesangial expansion is a hallmark of glomerulosclerosis. Examination of renal histopathological changes with PAS revealed that WD induced mesangial expansion which was blunted by INT-747 treatment to the level of the control group (Fig. 2F, G). In addition we used PDGFRβ as a mesangial cell marker to determine the mesangial area in the glomerular tuft by immunofluorescence imaging (18, 46). Although WD feeding caused increased mesangial area in DBA mice, INT-747 treatment ameliorated mesangial cell growth as shown by decreased PDGFRβ-positive area in glomeruli (Fig. 2H).

Fig. 2.

INT-747 treatment improves proteinuria and ameliorates renal structural changes in WD-fed DBA mice. A: urinary albumin excretion in DBA/2J mice, expressed as urinary albumin-to-creatinine ratio. B: podocin protein abundance was determined by Western blotting. C: immunofluorescence staining of kidney sections for podocin. D: immunofluorescence staining of kidney sections for synaptopodin. E: immunohistological detection of WT-1 in DBA/2J glomeruli. WT1 staining is indicated by intense immunoperoxidase activity in podocyte nuclei (arrows). Podocyte density is presented as numbers of podocytes per glomerular area. F: representative periodic acid-Schiff (PAS) staining of kidney sections (×400). G: mesangial expansion index is defined with the ratio of mesangial area to glomerular tuft area. The mesangial area is determined by assessment of the PAS-positive and nucleus-free area in the mesangium. H: relative mesangial area is shown as the ratio of mesangial area represented by the positive PDGFR-β staining area to the glomerular area. Representative data of 3 independent experiments with similar results are shown. *P < 0.05 vs. CON. **P < 0.05 vs. WD (n = 6 mice/group).

FXR activation by INT-747 promotes antifibrotic effects.

Glomerulosclerosis and tubulointerstitial fibrosis are the pathological hallmarks of progressive renal disease, characterized by an accumulation in the kidney of extracellular matrix proteins and myofibroblasts, primary matrix-producing cells (43). TGF-β is a cytokine that plays a pivotal role in the profibrotic responses (5, 6). The signal transduction of TGF-β is regulated via a heteromeric complex of type I and type II TGF-β receptors. The type II receptor is the primary binding protein for ligands and is the most important for signal transduction (1). In WD-fed DBA mice, we found a significant increase in type II TGF-β receptor expression in the kidney. The INT-747 treatment blocked the increase in type II TGF-β receptor expression, suggesting that FXR activation may counteract the TGF-β action in inducing kidney fibrosis (Fig. 3A). TGF-β is an important downstream mediator for the accumulation of extracellular matrix components. We therefore examined the expression of two extracellular matrix proteins, fibronectin and collagen IV. WD increased renal mRNA expression of fibronectin and protein expression of fibronectin and collagen IV in glomeruli and tubulointerstitial cells, which were significant inhibited by INT-747 treatment of WD-fed DBA mice (Table 2, Fig. 3, B and C). INT-747 showed the same inhibitory effect on the expression level of two myofibroblast markers, fibroblast-specific protein-1 (FSP-1) and α-SMA. INT-747 reduced FSP-1 mRNA expression and prevented the increase in α-SMA protein expression in the kidneys of WD-fed DBA mice (Table 2, Fig. 3D).

Fig. 3.

INT-747 treatment promotes antifibrotic effects. A: transforming growth factor-β type II receptor (TGFβR2) protein abundance was analyzed by Western blotting. B: immunofluorescence staining of kidney sections for fibronectin in glomeruli and tubulointerstitial area. C: immunofluorescence staining of kidney sections for collagen IV in glomeruli and tubulointerstitial area. D: INT-747 prevents the increase of α-smooth muscle actin (α-SMA) protein expression analyzed by Western blotting in the kidney of WD-fed DBA/2J mice. Representative data of 3 independent experiments with similar results are shown. *P < 0.05 vs. CON. **P < 0.05 vs. WD (n = 6–8 mice/group).

Table 2.

INT-747 treatment regulates renal gene expression

	DBA Control	DBA WD	DBA WD+INT-747
Extracellular matrix protein
Fibronectin	1.78±0.23	2.75±0.37*	1.29±0.03†
Kidney fibrosis marker
FSP-1	2.95±0.40	3.53±0.44	2.48±0.21†
Proinflammatory cytokines
MCP-1	1.65±0.15	3.75±0.89*	1.74±0.13†
Anti-inflammatory factors
KLF-2	1.43±0.13	1.24±0.11	2.19±0.44†
KLF-4	2.06±0.20	1.60±0.06*	3.24±0.58†
Thrombomodulin	2.14±0.18	1.77±0.19	3.35±0.34†
Oxidative stress
Nox-2	1.29±0.09	2.60±0.24*	1.81±0.12†
Fatty acid synthesis
SREBP-1c	4.10±0.55	8.04±1.07*	1.59±0.13†
ACC	1.57±0.08	1.59±0.07	1.19±0.03†
FAS	1.53±0.11	2.87±0.12*	1.25±0.13†
SCD-1	0.71±0.12	1.15±0.16	0.57±0.04†
Fatty acid oxidation and lipid catabolism
PPAR-α	2.23±0.15	1.09±0.06*	3.68±0.46†
CPT1a	1.20±0.02	0.84±0.04*	2.14±0.03†
UCP-2	1.29±0.03	1.36±0.08	2.99±0.19†
PGC-1α	2.40±0.08	2.36±0.07	4.00±0.08†
LPL	2.39±0.14	1.22±0.07*	1.63±0.06†

Values are means ± SE expressed as arbitrary units; n = 6–8 mice/group. Representative data of 3 independent experiments with similar results are shown. FSP-1, fibroblast-specific protein-1; MCP-1, monocyte chemotactic protein-1; KLF, Krüppel-like factor; Nox, NADPH oxidase; SREBP-1, sterol-regulatory element binding protein-1; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase, SCD-1, stearoyl-CoA desaturase-1; PPAR-α, peroxisome proliferator-activated receptor-α, CPT1a, carnitine palmitoyltransferase 1a; UCP-2, uncoupling protein-2; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; LPL, lipoprotein lipase.

^*P < 0.05 vs. control.

^†P < 0.05 vs. WD.

FXR activation by INT-747 modulates renal inflammation and oxidative stress.

Renal inflammation and oxidative stress are characteristic findings of both obesity and metabolic syndrome (35). In the present study, FXR activation showed a marked anti-inflammatory effect in WD-fed DBA mice. INT-747 treatment remarkably decreased mRNA expression of the proinflammatory chemokine monocyte chemotactic protein-1 (MCP-1) and induced mRNA expression of IκB-α, an inhibitory component of the nuclear factor-κB (NF-κB) signaling cascade (Table 2, Fig. 4A). The mRNA expression of p50 and p65 NF-κB heterodimeric complexes was not modified (data not shown). In contrast to MCP-1, the mRNA expression of anti-inflammatory factors Krüppel-like factor (KLF)-2, KLF-4, and their target gene thrombomodulin was significantly increased by INT-747 treatment (Table 2).

Fig. 4.

INT-747 treatment modulates renal inflammation and oxidative stress. A: mRNA expression level of IκB-α was determined by quantitative real-time PCR. B: protein abundance of manganese superoxide dismutase (MnSOD) was shown in Western blotting. Representative data of 3 independent experiments with similar results are shown. *P < 0.05 vs. CON. **P < 0.05 vs. WD (n = 6–8 mice/group).

FXR activation was also associated with antioxidative effects, as shown by decreased NADPH oxidase (Nox-2) expression in kidneys from INT-747-treated mice (Table 2). We have also examined the expression of MnSOD, the major antioxidant defense enzyme in the kidney. WD decreased the MnSOD protein level in the kidney, while INT-747 reversed this change, indicating its positive role in regulating oxidative stress (Fig. 4B).

FXR activation by INT-747 prevents renal triglyceride accumulation and regulates renal lipid metabolism.

Altered regulation of renal lipid metabolism has been shown to play an important role in the pathogenesis of diet-induced, obesity-related kidney disease (23, 25). In agreement with our earlier study in db/db mice with type 2 diabetes mellitus (22), we found that treatment with the FXR agonist significantly attenuates kidney triglyceride levels (Fig. 5). To explore the mechanism by which FXR activation regulates renal lipid metabolism, we investigated the effect of INT-747 treatment on pathways that regulate lipogenesis and lipolysis. We found that INT-747 reduces the mRNA expression of the fatty acid synthesis master gene SREBP-1c and its target genes acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase-1 (SCD-1) in the kidneys of WD-fed DBA mice (Table 2). We also found that INT-747 increased renal expression of peroxisome proliferator-activated receptor-α (PPAR-α), carnitine palmitoyltransferase 1a (CPT1a), uncoupling protein-2 (UCP-2), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and lipoprotein lipase (LPL) mRNAs, whose products are important for fatty acid oxidation and lipid catabolism (Table 2). Therefore, treatment with the FXR-activating ligand INT-747 inhibited the fatty acid synthesis pathways while stimulating fatty acid oxidation and lipid hydrolysis gene expression, resulting in decreased kidney triglyceride content.

Fig. 5.

INT-747 treatment regulates renal triglyceride content. Kidney triglyceride content was measured in lipid extracts from kidney homogenate samples. Representative data of 3 independent experiments with similar results are shown. *P < 0.05 vs. CON. **P < 0.05 vs. WD (n = 6–8 mice/group).

INT-747 regulates renal gene expression in an FXR-dependent manner.

To confirm the FXR dependency of the renal effects induced by INT-747, we treated FXR^−/− mice with INT-747 for 1 wk and compared renal gene regulation in wild-type mice on the same treatment. Previous studies from our laboratory have shown an acute regulation of genes involved in renal lipid metabolism by FXR agonist treatment of db/db mice with type 2 diabetes mellitus (22). Based on real-time PCR analysis, we have found that short-term INT-747 treatment decreases mRNA expression of SREBP-1c and SCD-1 and increases expression of UCP-2 and Nox-2 in wild-type C57BL/6 mice (Fig. 6). A similar regulation was observed in the WD-fed DBA mice. The same treatment in age- and sex-matched FXR^−/− mice failed to modify mRNA expression levels of these genes, indicating that the renal gene regulation induced by INT-747 is mediated by FXR-dependent signaling.

Fig. 6.

INT-747 regulates renal gene expression in an farnesoid X receptor (FXR)-dependent manner. Analysis of mRNA expression by quantitative real-time PCR for sterol-regulatory element binding protein-1c (SREBP-1c; A), stearoyl-CoA desaturase-1 (SCD-1; B), uncoupling protein-2 (UCP-2; C), and NADPH oxidase (Nox-2; D) shows the FXR-dependent regulation by INT-747. Representative data of 3 independent experiments with similar results are shown. *P < 0.05 vs. wild-type C57BL/6J mice (WT; n = 6 mice/group).

DISCUSSION

The purpose of this study was to examine the role and therapeutic efficacy of FXR activation in obesity-induced renal disease. In our DIO model, a Western-style diet induced in DBA/2J mice obesity, hyperglycemia, dyslipidemia, as well as hyperinsulinemia. In kidneys of DBA/2J mice fed with WD, glomerulosclerosis and tubulointerstitial fibrosis develop, characterized by proteinuria, podocyte injury, mesangial expansion, and accumulation of extracellular matrix proteins. Treatment with the selective FXR agonist INT-747 significantly ameliorates obesity-related renal injury by modulating renal lipid metabolism, fibrosis, inflammation, and oxidative stress.

Different mechanisms have been implicated in the induction of renal lesions associated with obesity, including renal lipid accumulation, inflammation, and oxidative stress (35). Our previous studies have shown that renal accumulation of lipids may play a role in the pathogenesis of diabetic nephropathy (20–23, 36, 44, 49). In the study of high-fat diet-induced obesity in C57BL/6J mice, we demonstrated that DIO causes renal lipid accumulation and glomerulosclerosis via an SREBP-1c-dependent pathway, indicating a new therapeutic strategy to prevent the obesity-induced renal disease through the modulation of renal lipid metabolism. Here we report, for the first time, treatment of this syndrome in a mouse model of DIO by a selective FXR agonist, INT-747.

INT-747 is a high-affinity, semisynthetic, bile acid-derived FXR agonist (33). It shows high bioavailability and limited off-target effects (45). By using FXR null mice, we have here confirmed the FXR-mediated effects on renal gene regulation by INT-747. Furthermore, unlike natural bile acids which can activate FXR-independent pathways via TGR5 (50), a G protein-coupled receptor functioning as a membrane bile-acid receptor, INT-747 does not induce, as predicted by its selectivity (50), kidney expression of TGR5 and its direct target gene type 2 iodothyronine deiodinase, a thyroid hormone activating enzyme (data not shown).

The DIO model was carried out in DBA/2J mice, a strain more prone to diabetic nephropathy compared with the widely used C57BL/6J mice, which are relatively resistant to development of this pathology (37). In the current study, we have observed a progressive weight gain in WD-fed DBA/2J mice which was blunted by INT-747, an effect becoming apparent within 4 wk after treatment initiation. This contrasts with the lack of efficacy shown by GW4064 in the C57BL/6J mouse DIO model, which did not reveal any protective effect against weight gain (50). This discrepancy may reflect the different bioavailability of these two FXR agonists (45). In this regard, a cholic acid diet has been shown to completely prevent and reverse diet-induced obesity via a TGR5-dependent mechanism (50). INT-747 has also demonstrated a lipid-lowering effect in our DIO model by reducing both plasma HDL-C and LDL-C. Although HDL-C/TC was not changed, the proatherogenic LDL-C/TC was decreased by the treatment. This finding is consistent with the reported antiatherosclerotic effects of INT-747 (16, 26).

There is growing evidence for the role of dysregulated lipid metabolism in the pathogenesis of renal disease (20–23, 25, 35, 36, 38, 44, 49). Consistent with our previous findings (22, 23), FXR activation prevents dietary fat-induced increases in renal expression of SREBP-1c and its target genes, as shown here, providing further evidence that accelerated renal lipogenesis contributes to renal lipid accumulation in the pathogenesis of nephropathy. Furthermore, our present study demonstrates that INT-747 enhances expression of renal genes involved in lipolysis and fatty acid oxidation, including LPL, PPAR-α, and PGC-1α. LPL increases hydrolysis of triglycerides into free fatty acids, whereas PPAR-α induces the expression of a number of enzymes involved in peroxisomal and mitochondrial fatty acid β-oxidation. PGC-1α can also regulate genes involved in fatty acid catabolism through its coactivation of PPAR-α. Collectively, our data suggest a novel mechanism by which FXR activation elicits coordinated actions of broad transcriptional regulators such as SREBP-1c, PPARs, and PGC-1α, thus decreasing renal content of lipids and minimizing lipotoxicity.

Inflammatory abnormalities are characteristic findings of both obesity and metabolic syndrome (35), as also shown in our DIO model by the markedly increased proinflammatory cytokine expression. Wu et al. (53) found increased expression of genes related to lipid metabolism, inflammatory cytokines, and insulin resistance in glomeruli of patients with obesity-related glomerulopathy compared with controls. In addition, the phenotype of Zucker fatty/spontaneously hypertensive heart failure (ZSF) rats with severe metabolic syndrome and overt nephropathy is characterized by abnormal renal lipid drops and severe capillary infiltration of polymorphonuclear neutrophils and macrophages (10). These findings strengthen a role for inflammation in obesity-related kidney disease, as a consequence of tissue lipid accumulation or secondary to by-products of lipid metabolism. This view is further supported by a study showing the efficacy of the immune suppressant mycophenolate mofetil in limiting renal injury in obese ZSF rats without altering the severe lipotoxic renal phenotype (9).

A novel finding of our study is the capacity of FXR agonists like INT-747 to upregulate IκB-α, KLF-2, and KLF-4 expression. Anti-inflammatory properties of FXR were appreciated in many studies (15, 22, 26, 30). FXR has been shown to negatively interfere with the inflammatory response by antagonizing the NF-κB signaling pathway (26). NF-κB transcriptional activity controls the activation of a number of inflammatory genes, including MCP-1. However, the detailed molecular mechanisms have yet to be elucidated. Here, our data provide evidence for potential mechanisms underlying overall anti-inflammatory activities of FXR-dependent signaling. First, the induction of IκB-α by INT-747 could affect the p65 nuclear translocation or reduce NF-κB DNA-binding activity by the acceleration of NF-κB nuclear deactivation. Interestingly, this mechanism is shared by other nuclear receptors, like PPARα or VDR, whose activation by ligand induces IκB-α expression, contributing to the anti-inflammatory properties (8, 13). Second, our data suggest a function for KLFs involved in the anti-inflammatory activity of FXR in obesity-related renal disease. The KLF family of transcription factors has been implicated in several biological processes, including proliferation, apoptosis, differentiation, and development (3). The function of KLF family members in nephropathy has never been explored before. KLF-2 and KLF-4 act as antiatherosclerotic and anti-inflammatory regulators of endothelial activation in response to proinflammatory stimuli. KLF2 inhibits endothelial inflammation via multiple distinct mechanisms that inhibit the NF-κB pathway (7, 32, 40), suggesting that FXR can antagonize NF-κB signaling by targeting multiple pathways involved in its regulation.

In summary, our findings identify the effect of FXR activation, using a selective and potent FXR agonist, on the prevention of obesity-related renal pathology and on potential metabolic pathways that may mediate obesity-induced alterations in renal lipid metabolism, fibrosis, inflammation, and oxidative stress. This knowledge has translational potential into effective therapies for patients with kidney pathology associated with obesity.

GRANTS

The work was supported by National Institutes of Health Grants U01 DK076134 and R01 AG026529, the Juvenile Diabetes Research Foundation, and a Veterans Affairs Merit Review.

DISCLOSURES

L. Adorini and M. Pruzanski are with Intercept Pharmaceuticals. The other authors declared no competing interests.