Obesity is not only the major risk factor for cardiovascular disease but also a main cause of renal disease ((1), (2)). Abdominal obesity is the leading risk factor for albuminuria. Weisinger reported the first case of obesity‐induced kidney impairment in 1974, naming it obesity‐related glomerulopathy. Later studies found that obesity‐related glomerulopathy had obvious distinctions from primary glomerulosclerosis because of its special pathologic characteristics, which include hypertrophy and glomerulosclerosis with a series of metabolic comorbidities. Severe obesity is associated with increased systemic arterial pressure ((3)), high renal plasma flow ((4), (5)), increased glomerular filtration rate (GFR) ((4)), and an increased albumin excretion rate ((6)). In addition to these physiologic abnormalities, obesity has been associated with the nephrotic syndrome and renal failure ((7), (8)). Visceral fat accumulation contributes to metabolic abnormalities and inflammation ((9)). Increased blood levels of insulin and lipids as well as increased inflammation are all responsible for the visceral fat‐derived renal impairment; however, the signaling pathway involved in the fat‐derived renal impairment has not been fully elucidated.
Recent studies showed peroxisome proliferator‐activated receptors (PPARs) play important roles in the metabolic syndrome and its related renal complications. PPARα in the proximal tubules plays an important role in the metabolic control of renal energy homeostasis ((10)). PPARγ and PPARα agonists are beneficial in diabetic nephropathy ((11), (12)). PPARδ regulates multiple proinflammatory pathways to suppress atherosclerosis ((13)). PPARγ agonists have been used to treat type 2 diabetes and diabetic nephropathy; however, specific drugs used to treat obesity‐related glomerulopathy are lacking because of unclear mechanisms. PPARδ, another isoform of PPAR, was recently shown to have various roles in lipid regulation, adipocyte differentiation, and cell proliferation. Targeted activation of PPARδ in adipose tissue reduced adiposity ((14)). PPARδ is not only associated with adipocyte differentiation and fat storage but also correlated with inflammation, glucose metabolism, and lipid metabolism ((15)). In addition to being expressed in adipose tissue, PPARδ is also expressed in the collecting duct, glomerulus, and microvasculature of the kidney. A previous study showed PPARδ exerts a strong protection from acute ischemic renal failure by activating the antiapoptotic Akt signaling pathway ((16)). In mice with type 1 diabetes, lower renal PPARδ expression results in renal lipotoxicity because of reduced fatty acid oxidation ((17)). The PPARδ activation shows renoprotective effects through its anti‐inflammatory activity by inhibiting MCP‐1 expression and macrophage infiltration in the diabetic kidney ((18)).
Mesangial proliferation is a key feature in the pathogenesis of a number of renal diseases and can be experimentally induced by the mitogen platelet‐derived growth factor (PDGF). PPARγ ligand attenuates PDGF‐induced mesangial cell proliferation through p38 Mitogen‐activated protein kinase (p38 MAPK) inhibition ((19)). MAPK signaling plays a key role in nondiabetes and diabetes nephropathy through regulating mesangial cell proliferation. Thus, we hypothesized that glomerular mesangial cell proliferation, extracellular matrix (ECM) accumulation, and finally, obesity‐related glomerulopathy might be induced by PPARδ suppression and p38 MAPK activation. To test this hypothesis, we explored the roles of PPARδ and p38 MAPK in the obesity‐related glomerulopathy in high‐fat‐diet‐induced rat model of metaboic syndrome.
Methods and Procedures
Animals and experimental procedures
All of the experimental procedures were approved by the Institutional Animal Care and Research Advisory Committee. Male Wistar rats obtained from an in‐house breeding colony were randomized into two groups at 2 months of age; one group received standard laboratory chow ad libitum and the other group received a safflower oil‐based high‐fat diet ad libitum, as previously described ((20), (21)). The high‐fat diet supplied 59% of the calories as fat and 20% of the calories as carbohydrate comprising cornstarch and sucrose (2:1 w/w). The chow diet provided 10% of the calories as fat and 65% as carbohydrate. The chow or high‐fat diets were provided for 32 weeks. All animals had free access to water and were subject to controlled temperature (22 ± 1°C) and lighting (lights on from 06.00 to 18.00). Systolic blood pressure and diastolic blood pressure were measured using an interventional method (model MLT 1030, Power Lab, AD Instruments). Body weight and blood glucose were measured every week. Insulin sensitivity was assessed using the euglycemic‐hyperinsulinemic glucose clamp technique. At the end of 32 weeks, a 24‐hour urine sample was collected in a metabolic cage to measure protein and micro‐albuminuria. Blood lipid levels, fasting blood glucose, and fasting insulin were detected using commercially available kits. Urinary albumin excretion rate (UAER) was determined using a radioimmunoassay. The rats were killed by decapitation. Kidney tissue was removed and used for measurements. Visceral fat was removed and weighed. Glucose infusion rate was calculated using the following formula: glucose infusion rate (GIR) (mg/kg/min) = Volume (ml/kg/d) × glucose concentration (as a decimal)/1.44.
The kidney was cleaned with saline and weighed. It was then fixed in buffered formalin saline for Periodic acid‐Schiff (PAS) staining to evaluate the degree of renal lesions. The PAS‐stained kidney sections were examined under a light microscope with a 20× objective lens. Five glomeruli were measured in each section, and the average was calculated using NIS‐Elements 3.2 (Nikon Corporation) imaging software. The glomerular matrix and glomerular area ratio were also calculated to obtain the relative area of the glomerular ECM. Glomerular volume was calculated using the formula, GV = β/κ (GA)3/2, where β = 1.38 and represents the shape coefficient for a sphere, κ = 1.1, and represents the size distribution coefficient and GA is glomerular cross‐sectional area ((22)). The remaining tissue was placed in phosphate‐buffered saline for the following experiments.
PPARδ and phosphorylated p38 MAPK immunohistochemistry
For immunohistochemistry, tissue samples were fixed in 4% buffered paraformaldehyde and embedded in paraffin. Four‐micrometer sections were cut from paraffin‐embedded tissue samples and mounted on poly‐L‐lysine‐coated slides. Slides were fixed overnight at 56°C in an incubator. Sections were deparaffinized and rehydrated. After 2 min of washing with distilled water, slides were treated for 15 min with a 3% hydrogen peroxide solution in PBS to block endogenous peroxidase activity. Tissue sections were washed three times for 3 min in PBS‐Tween and incubated for 30 min with anti‐PPARδ or anti‐phosphorylated p38 MAPK (p‐p38 MAPK) monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS/1% BSA. Sections were rinsed three times for 3 min in PBS‐Tween and incubated for 30 min with peroxidase‐conjugated polymer, which also carries antibodies to mouse immunoglobulins (Dako Cytomation, Copenhagen, Denmark). After three cycles of washing, sections were incubated with diaminobenzidine (DAB) for 10 min and then rinsed with distilled water. Tissue sections were dehydrated and covered with Pertex and a coverslip. Images were recorded and analyzed using NIS‐Elements 3.2 software (Nikon Corporation). Immunoperoxidase staining was evaluated in all images by application of a threshold procedure to highlight the positive areas selectively. The software automatically calculated the percentage of the stained area.
Coculture and treatment of glomerular mesangial cells and mature 3T3‐L1 cells
Glomerular mesangial cell strain HBZY‐1 was acquired from the CCTCC (China Center for Type Culture Collection, Wuhan, China). Cells were grown in Dulbecco's modified Eagle's medium (DMEM; GIBCO‐BRL, Grand Island, NY) containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mmol/l glutamine in an atmosphere of 5% CO2 and 95% air at 37°C. 3T3‐L1 preadipocytes were cultured and differentiated into matured adipocytes as previously described ((23)). Murine 3T3‐L1 preadipocytes as previously described were cultured and maintained in DMEM supplemented with 10% fetal calf serum (HyClone) containing 100 μg/ml penicillin and 100 μg/ml streptomycin (GIBCO). Cells were plated and grown until 2 days post‐confluence. Differentiation was then induced (day 0) by changing the medium to DMEM supplemented with 10% fetal calf serum, 0.5 mmol/l 3‐isobutyl‐L‐methylxanthine, 1 μmol/l dexamethasone, and 5 μg/ml insulin. Mature differentiation 3T3‐L1 cells were fixed and stained with the lipophilic dye oil red O (Sigma‐Aldrich). Red staining shows lipid droplets in the cytoplasm indicating adipocyte differentiation.
The coculture system of mature differentiation 3T3‐L1 and HBZY‐1 was established in 12‐well Corning Transwell plates (Fisher), a noncontact coculture system, with two compartments separated by a polycarbonate membrane with 0.4 μm pores. In some experiments, HBZY‐1 cells were transfected with PPARδ‐expressing or empty vectors in the lower compartment, and then mature differentiation 3T3‐L1 cells were added to the upper compartment for 48 h. In some experiments, HBZY‐1 and mature 3T3‐L1 cells were cocultured for 24 h and then treated with a PPARδ inhibitor, GSK 0660 (5 μM, Sigma), in the presence or absence of a p38 MAPK inhibitor, SB239603 (1 μM, Sigma), for another 24 h. Protein levels were detected in HBZY‐1 cell homogenates by Western blotting. Concentrations of laminin and type IV collagen were assayed in the culture medium by radioimmunoassay using commercially available assay kits.
Overexpression of PPARδ in HBZY‐1 cells
The recombinant adenoviral vectors containing rat PPARδ were generated as described ((24)). The cDNA encoding rat PPARδ was amplified by RT‐PCR using the isolated total RNA as the template from the white fat. The recombined shuttle plasmid, pAdTrack‐CMV‐PPARδ, was constructed by linking the pAdTrack‐CMV with PPARδ; the plasmid was then sequenced. To generate recombinant adenoviral plasmids, we linearized pAdTrack‐CMV‐PPARδ with PmeI and transformed into electrocompetent Escherichia coli BJ5183 cells, which contained adenoviral backbone vectors (pAdEasy‐1). Clones with inserts were screened by restriction endonuclease digestion. Once confirmed, supercoiled plasmid DNA was transformed into LX10 cells for large‐scale amplification. To produce adenoviruses in AD293 cells, a transfection mix was prepared by adding 4 μg of PacI linearized plasmid DNA, which was purified by gel extraction and 20 μl of Lipofectamine (Life Technologies) to 500 μl of OptiMEM (Life Technologies). Transfected cells were monitored for green fluorescent protein expression. After 7‐10 days, transfected cells were collected. After three cycles of freezing in a methanol dry ice bath and rapid thawing at 37°C, viral lysate was used to infect AD293 cells in flasks. Three to four days later, viruses were harvested as described above. After repeating this process several times, viral titers were often high enough to use for gene transfer experiments in cultured cells.
Western blot analysis
Immunoblotting of PPARδ, p‐p38 MAPK, and GAPDH was performed using standard techniques as we previously described ((25)). Primary antibodies from Santa Cruz Biotechnology were used for kidney tissue and glomerular mesangial cells. After incubation with the secondary antibodies for 1 h, the proteins were detected by enhanced chemiluminescence and quantified using a Gel Doc 2000 Imager (Bio‐Rad). The immunoblots showed a band of the expected size for PPARδ and p‐p38 MAPK. Experiments were performed in three independent experiments.
Data are presented as the mean ± SEM of n experiments. Significant differences between groups were assessed using Student's t‐test or one‐way ANOVA with Bonferroni's multiple comparison post hoc tests as appropriate. Two‐sided P‐values < 0.05 were considered statistically significant.
High‐fat‐diet‐induced metabolic and renal dysfunction in rats
We used a rat model of metabolic syndrome. We analyzed the biometrical and biochemical characteristics of rats on a normal diet and a high‐fat diet (HFD). Table 1 shows the biometrical characteristics of the control group and the HFD group. After administration for 32 weeks, rats in the HFD group had significantly higher body weight, blood pressure, visceral fat mass, and kidney weight compared with that in the control group (Table 1, Figure 1a‐c). Compared to the control group, the HFD group had higher levels of triglycerides and free fatty acids (Table 1). Rats on a HFD also had more severe insulin resistance. A high plasma insulin‐normal glucose clamp test showed that the HFD group had significantly higher fasting insulin levels and a lower GIR (Table 1). Microalbuminuria was assessed to evaluate renal function. As shown in Figure 1d, UAER in the HFD group was significantly higher than that in control group, suggesting impaired renal function.
Table 1. General characteristics of ratsControlHFDN1111SBP (mmHg)126 ± 2151 ± 4**DBP (mmHg)81 ± 2105 ± 4**Body weight (g)510 ± 24704 ± 22**FBG (mmol/L)5.21 ± 0.366.11 ± 0.31**FFA (mmol/L)2.24 ± 0.193.01 ± 0.14**TG (mmol/L)1.32 ± 0.041.56 ± 0.09**FINS (μU/mL)25.62 ± 4.7866.04 ± 7.23**GIR (mg/kg/min)1.51 ± 0.186.10 ± 0.34**
HFD, high‐fat diet; SBP, systolic blood pressure; DBP, diastolic blood pressure; FBG, fasting blood glucose; FFA, free fatty acids; TG, triglycerides; FINS, fasting insulin; GIR, glucose infusion rate. Data are mean ± SEM, **P < 0.01 vs. Control.
Biometrical and biochemical characteristics of rats on a normal diet and a high‐fat diet (HFD). (a) Weight of kidney; (b) ratio of kidney weight to body weight; (c) weight of visceral fat; (d) urinary albumin excretion rates (UAER). Data are presented as the mean ±SEM for each group (11 rats per group). *P < 0.05, **P < 0.01 vs. control.
Glomerular hypertrophy and expression of PPARδ and p‐p38 MAPK in rat kidney
First, we observed that rats on a HFD had significantly increased glomerular volume (Figure 2a, b), a thickened basement membrane, a smaller Bowman's capsule, a thickened capsule wall, and expanded renal tubules (Figure 2a). There was also significantly more accumulation of glomerular ECM in the HFD group (Figure 2c). These data indicate that the HFD caused glomerular hypertrophy. We then investigated the expression of PPARδ p‐p38 MAPK in renal tissues using immunohistochemistry. Recent studies indicated that the PPARδ agonist, GW0742, attenuates renal dysfunction, which is accompanied by a significant reduction in medullary necrosis, apoptosis, and inflammation ((16), (26)). P38 MAPK has been implicated in mediating mesangial cell contraction in response to several vasoactive factors, including angiotensin II, related to the development of diabetic nephropathy ((27), (28)). Our results showed that PPARδ and p‐p38 MAPK both exist in glomerular cells. Compared to the control group, PPARδ expression was strongly decreased in the HFD group (Figure 2a, d), whereas p‐p38 MAPK was significantly increased (Figure 2a, e). We also measured the expression of PPARδ, phosphorylation of p38 MAPK, and total p38 MAPK in renal tissues using Western blotting. The results showed that the HFD reduced PPARδ expression by 18% compared to the control diet (0.50 ± 0.02 vs. 0.61 ± 0.04, P < 0.05, Figure 2f). Phosphorylation of p38 MAPK to the total p38 MAPK expressions increased by 62% in rats on HFD compared to the control diet (1.59 ± 0.15 vs. 0.98 ± 0.14; P < 0.01), but the total p38 MAPK was not different between these two groups (P > 0.05, Figure 2g). These data indicate that the glomerular hypertrophic responses in rats on a HFD are associated with reduced PPARδ expression and increased p38 MAPK phosphorylation.
Glomerular histology and expression of PPARδ and p‐p38 MAPK in the kidneys of rats on a control diet and a high‐fat diet (HFD). (a) Representative microscopy (magnification ×400) of glomerular histology shown by periodic acid‐Schiff (PAS) staining and immunohistochemistry for PPARδ and p‐p38 MAPK; (b) glomerular volume; (c) glomerular ECM accumulation; (d) fraction of area that stained positively for PPARδ; (e) fraction of area that stained positively for p‐p38 MAPK; representative immunoblots and summarized data of PPARδ (f) and p‐p38/p38 MAPK (g) protein levels in the kidney. Data are presented as the mean ± SEM from 4 to 10 separate experiments. *P < 0.05, **P < 0.01 vs. control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
PPARδ inhibits laminin and collagen production via suppression of p38 MAPK phosphorylation in mesangial cells
To explore the mechanism of obesity‐related glomerulopathy and the relationship between PPARδ and p38 MAPK, we cultured and transfected HBZY‐1 cells with PPARδ or treated with PPARδ agonist GW 0742. We found that phosphorylation of p38 MAPK but not total p38 MAPK level was significantly reduced by transfected with PPARδ (Figure 3a, b) or treated with GW 0742 (Figure 3e, f). Furthermore, Laminin and collagen IV production were also significantly inhibited after being transfected with PPARδ (Figure 3c, d) or treated with GW 0742 in HBZY‐1 cells (Figure 3g, h), and these effects were absence in HBZY‐1 cells transfected with empty vectors. Furthermore, we treated HBZY‐1 cells with a PPARδ antagonist, GSK0660, with and without a p38 MAPK antagonist, SB239603, and then examined p‐p38 MAPK and total p38 MAPK, laminin, and collagen IV levels. p‐p38 MAPK but not total p38 MAPK was significantly increased in HBZY‐1 cells by the PPARδ antagonist, GSK0660, whereas this increase was almost completely abolished in the presence of the p38 MAPK antagonist, SB239603 (Figure 4a, b). Similarly, laminin and collagen IV production were markedly increased by GSK0660, whereas this increase was significantly inhibited in the presence of SB239603 (Figure 4c, d). These results strongly indicate that PPARδ suppression promotes laminin and type IV collagen secretion through p38 MAPK phosphorylation in mesangial cells.
PPARδ overexpression or agonist GW 0742 attenuates phosphorylation of p38 MAPK and prevents laminin and type IV collagen secretion. (a) Representative immunoblots and summarized data; (b) of p‐p38/p38 MAPK protein levels in HBZY‐1 cells. Concentration of LN (c) and Col IV (d) in the coculture medium from HBZY‐1 and mature differentiation 3T3‐L1 cells. Data are presented as the mean ± SEM from three separate experiments. *P < 0.05, **P < 0.01 vs. control; ▴P < 0.05, ▴▴P < 0.01 vs. empty vector. (e) Representative immunoblots and summarized data of p‐p38/p38 MAPK; (f) protein levels in HBZY‐1 cells. Concentration of LN (g) and Col IV (h) in the coculture system medium from mature differentiation 3T3‐L1 and HBZY‐1. Data are presented as the mean ± SEM from three separate experiments. *P < 0.05 vs. control.
P‐p38 MAPK level and laminin (LN) and type IV collagen (Col IV) productions in HBZY‐1 cells treated with PPARδ inhibitor GSK 0660 in the presence or absence of p38 MAPK inhibitor SB239603. (a) Representative immunoblots and summary data; (b) of p‐p38 MAPK levels in HBZY‐1 cells. Concentration of LN (c) and Col IV (d) in the coculture system medium from HBZY‐1 and mature differentiation 3T3‐L1 cells. Data are presented as the mean ± SEM from 4 separate experiments. *P< 0.05, **P < 0.01 vs. control; ▴P < 0.05, ▴▴P < 0.01 vs. GSK0660.
In this study, the novel finding is that glomerular hypertrophy was associated with decreased PPARδ expression and elevated phosphorylation of p38 MAPK in rats on HFD‐induced metaboic syndrome. PPARδ transfection inhibits laminin and type IV collagen secretion through p38 MAPK suppression in mesangial cells.
Rats on a long‐term HFD showed typical characteristics of metabolic syndrome, including severe obesity, dyslipidemia, insulin resistance, and hypertension. These findings confirmed earlier results reported by Chalkley et al ((20)). Obesity is one of the prominent components of metabolic syndrome. Obesity‐related glomerulopathy is always accompanied by various metabolic disorders in metabolic syndrome, which eventually leads to end‐stage renal diseases. The rat model of metabolic syndrome induced by a long‐term HFD can be used effectively to study the pathology of obesity and metabolic syndrome‐related glomerulopathy.
Few studies exmained the effect of HFD or metabolic syndrome on PPARδ level in renal glomerulus. However, metabolic syndrome accelerates inflammation in kidney tissues ((10)). Activation of PPARδ down‐regulates the expression of the receptor for advanced glycation end products ((29)) and inhibits mouse diabetic nephropathy through anti‐inflammatory mechanisms ((18)).
P38 MAPK signaling plays a distinct pathogenetic role in the progression of nephropathy in animal models of diabetes and obesity ((28), (30)). In diabetic Otsuka Long‐Evans Tokushima Fatty rats, the p38 MAPK pathway was activated in renal tissue and the inhibition of p38 MAPK led to decreased urinary protein excretion ((30)). Activation of mesangial cell MAPK subgroups by hypertrophic stimuli has also been documented ((31)). PPARδ is a widely expressed transcription factor regulating lipid and glucose metabolism, inflammation, adipocyte differentiation, and cell proliferation. The expression of PPARδ in the kidney might be beneficial in response to obesity and metabolic disorders. A previous study showed that PPARδ exerted a strong protection from acute ischemic renal failure ((16)). Oxidative stress via ROS has been implicated in renal inflammation of metabolic syndrome ((32)). ROS may participate in p38 MAPK regulation ((33)). Activation of PPARδ resulted in an NF‐κB‐dependent transcriptional and posttranscriptional MAPK signaling pathway in endothelial cell ((34)). In this study, PPARδ expression was reduced and p‐p38 MAPK was increased in rats with obesity, metabolic syndrome, and glomerular hypertrophy. Thus, PPARδ suppression and p38 MAPK activation might play critical roles in the progression of obesity‐related glomerulopathy.
We further explored the molecular mechanism of obesity‐related glomerulopathy in a coculture system of mesangial cells and preadipocytes. PPARδ suppression promoted laminin and type IV collagen secretion through p38 MAPK phosphorylation in mesangial cells, whereas PPARδ overexpression attenuated p38 MAPK phosphorylation and laminin and type IV collagen secretion. These in vitro findings highlight the pathogenetic role of PPARδ in obesity‐related glomerular hypertrophy, which is partly consistent with previous reports suggesting an involvement of PPARδ in metabolic kidney disease. PPARδ plays an important role in renal metabolic adaptation to fasting and refeeding ((35)). In mice with type 1 diabetes, renal expression of PPARδ is greatly suppressed, which may contribute to renal lipotoxicity ((17)). Treatment of mesangial cells with IGF‐1, a cytokine upregulated in the diabetic kidney ((36)), increased triglyceride accumulation, possibly via PPARδ suppression ((37)). A recent study showed that PPARδ provided strong protection against ischemia‐induced renal injury as a result of its combined action on cell survival and cytoskeletal reorganization ((16)).
Previous studies have not given conclusive information on the effects of PPARδ on p38 MAPK and cell proliferation. Several studies reported that PPARδ promoted cell proliferation through cooperation with MAPK signaling ((38), (39)); however, this study demonstrated that PPARδ inhibits p38 MAPK activation and thus attenuates mesangial cell laminin and collagen IV secretion, an important characteristic of glomerular hypertrophy. This result is partly in accordance with a previous report showing that the PPARδ ligand, GW501516, reduces growth but not apoptosis in mouse inner medullary collecting duct cells ((40)).
In conclusion, our study describes the characteristics of glomerulopathy in rats with obesity and metabolic syndrome and reveals a novel pathogenetic mechanism. Enhanced p38 MAPK activation and laminin and collagen IV secretion induced by PPARδ suppression in mesangial cells might contribute to the progression of obesity‐related glomerular hypertrophy. This might provide new targets for the clinical treatment of obesity and metabolic syndrome‐related glomerulopathy.
Zhencheng Yan, Yinxing Ni, Peijian Wang, Jian Chen, Hongbo He, Jing Sun"Peroxisome proliferator‐activated receptor delta protects against obesity‐related glomerulopathy through the P38 MAPK pathway" Obesity Volume21, Issue3 March 2013