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Introduction

Recent statistics from the Centers for Disease Control, the National Institutes of Health and the World Health Organization report that obesity has more than doubled since 1980, and almost two billion adults worldwide are overweight or obese (1). Obesity is a preventable disease; however, it is evident that current educational efforts have failed to counteract its upward trend. The growing epidemic of obesity in the world is a major factor in reducing life expectancy, and represents an economic burden and a serious health problem. It is largely responsible for the growing incidence and prevalence of diabetes, as well as cardiovascular and renal disease. Evidence indicates that obesity is a risk factor for the development of renal inflammation and dysfunction, that may progress towards chronic kidney disease (CKD) (2,3).

Considering that obesity is associated with a chronic low-grade inflammatory condition, and that studies have demonstrated that obesity-induced inflammation develops in the renal tubularinterstitium (4), exploring the mechanisms of the renal tubulointerstitial inflammatory response in obesity may be of particular importance. Multiple lines of evidence suggest that the direct action of monocyte chemoattractant protein-1 (MCP-1) is a powerful instigator of tubulointerstitial inflammation (5,6). Excessive MCP-1 in the renal interstitium may recruit macrophages and generate inflammation, contributing to tubulointerstitial injury and the progression of CKD. Initially, circulatory MCP-1, produced and secreted by adipose tissue, had been the focus of attention as the root of tubulointerstitial inflammation. However, it has been reported that the concentrations of serum and urinary MCP-1 are not associated with each other in obesity, indicating a role for locally produced MCP-1 in the kidney (7). Therefore, research into the mechanisms of renal MCP-1 generation may be a useful step in understanding and developing novel treatments for human renal tubulointerstitial inflammation.

A previous study demonstrated that MCP-1 expression in the vascular smooth muscle cells of diabetic mice is negatively regulated by the transcription factor forkhead boxO1 (FOXO1) (8). Other reports have indicated that FOXO1 is able to regulate certain biological processes, including cell cycle arrest, DNA damage repair and oxidative stress inhibition (9–11). It has beneficial effects on renal injury by attenuating oxidative stress and inhibiting the activation of transforming growth factor beta-1 (TGF-β1) signaling in diabetic rats (12,13). However, whether FOXO1 is able to regulate MCP-1 production, thus mediating renal tubulointerstitial inflammation during obesity, is poorly understood. Furthermore, evidence shows that FOXO1 is a downstream target of mammalian target of rapamycin (mTOR) in human tracheal smooth muscle cells. When mTOR is activated by phosphorylation (p-mTOR), it stimulates FOXO1 phosphorylation (p-FOXO1) and translocation from the nucleus to the cytosol. In this case, FOXO1 is dissociated from the promoter of the target gene, enabling the gene to be expressed (14). Recent evidence demonstrates that mTOR serves a key role in the generation and development of renal inflammation, through functions such as NLRP3 inflammasome activation (15), and increasing autophagy in macrophages and renal cells (16), which mediates renal inflammatory cytokine release. Since mTOR is activated in obesity (17–19), the authors propose a possible scenario in which activated mTOR may enhance the phosphorylation of FOXO1, with p-FOXO1 subsequently dissociating from the MCP-1 promoter, followed by the increased expression of MCP-1, ultimately leading to a renal tubulointerstitial inflammatory response.

In the present study, renal function was initially evaluated in the participants with obesity, and the pathomorphology and associated mechanisms of tubulointerstitial inflammation were assessed in the high fat diet (HFD)-induced obese rat model and a cultured human renal tubular epithelial cell line (HK-2). The purpose of the current study was to provide insights into the onset of obesity-associated renal tubulointerstitial inflammation and to identify a potential novel approach to treating renal injury.

Materials and methods

Clinical study

A total of 35 subjects with obesity (19 males and 16 females) were recruited consecutively at the First Affiliated Hospital of Soochow University (Suzhou, China). The inclusion criteria were as follows: i) Obesity (BMI≥27 kg/m2); and ii) Chinese adults (including mainland China, Taiwan, and individuals from Hong Kong living in Jiangsu) aged 18–65. The exclusion criteria were as follows: i) Diabetes mellitus; ii) currently taking drugs that might interfere with the study, such as statins, fibrates, steroids, antihypertensive or anti-diabetic agents; iii) interstitial pneumonia; iv) severe renal or liver disease; v) malignant tumors; and vi) a history of acute coronary syndrome, stroke or acute pancreatitis within 3 months prior to enrollment. In addition, 27 normal weight, healthy subjects (15 males and 12 females) with a BMI<24 kg/m2 served as a control group. The Ethics Committee of the First Affiliated Hospital of Soochow University approved this study, and all subjects provided written informed consent. During the physical examination, body weight (BW), height and waist circumference (WC) were determined. Blood pressure (BP) was measured on the right arm using a standard mercury sphygmomanometer with the subject in a seated position after 10 min of rest. Venous blood samples were collected from overnight fasted subjects. Blood urea nitrogen (BUN), creatinine (Cr), total triglycerides (TG), total cholesterol (TC) and free fatty acid (FFA) were measured using a chemistry analyzer (Hitachi 7600; Hitachi, Ltd., Tokyo, Japan) at the central laboratory of the hospital. Urinary MCP-1 (u-MCP) and urinary neutrophil gelatinase-associated lipocalin (u-NGAL) concentrations were determined using ELISA kits (cat. no. ab179886, Abcam, Cambridge, UK; and cat. nos. SCB388Hu and SEB388Ra, Cusabio, Wuhan, China) according to the manufacturer's instructions. Urinary albumin was measured via the Coomassie brilliant blue method (cat. no. CO35-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The urinary albumin to creatinine ratio (UACR) was calculated from first voided spot samples using clean-catch techniques and sterile containers; ratios <30 mg/g creatinine were defined as normoalbuminuria, those between 30 and 300 mg/g creatinine as microalbuminuria, and those ≥300 mg/g creatinine as macroalbuminuria.

Animal model

Male Sprague-Dawley (SD) rats (5 weeks old; SIPPR-Bk Laboratory Animals Co., Ltd., Shanghai, China) weighing approximately 160 g were housed in polypropylene cages and maintained under controlled room temperature (22±2°C) and humidity (60±5%) with a 12 h light/dark cycle. Housing and animal experiments were approved by the Committee for Animal Subjects at Soochow University according to institutional and national animal welfare guidelines. After one week of adaptation, rats were randomly assigned to receive a normal chow diet (NCD; D12102C; Research Diets, Inc., New Brunswick, NJ, USA) or a HFD (60% kcal in fat; D12492; Research Diets, Inc.) or HFD plus 1 mg/kg/day of specific mTOR inhibitor rapamycin (Rap; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) by intraperitoneal injection (HFD+Rap; n=5 rats per group) for 8 weeks (20,21). A 24 h urine sample was collected from the rats after 3 days of adaptation in individual metabolic cages to measure 24 h urine protein and u-NGAL levels. The rats were then weighed and sacrificed. Blood was collected to test Cr, BUN, TG, TC and FFA.

Renal histology

The kidneys were perfused with saline solution and then removed and weighed. Kidneys were fixed in 10% neutral formalin for 24 h at 4°C and embedded in paraffin. Sequential paraffin-embedded tissue sections from the renal cortex were cut. Cross-sections (3 µm) were placed on gelatin-coated slides and used for hematoxylin-eosin (HE) staining, periodic acid-silver methenamine (PASM) staining and immunohistochemical staining for CD68 (a macrophage marker). In each kidney, at least 20 non-overlapping cortical areas of tubulointerstitial section were analyzed. The remaining kidney tissue was snap-frozen in liquid nitrogen and stored at −80°C for western blot analysis.

HE staining

The paraffin sections were stained using hematoxylin (cat. no. D006; Nanjing Jiancheng Bioengineering Institute) at room temperature for a total of 10 min, followed by washing under running water for 30–60 sec. The sections were then placed in eosin (cat. no. D006; Nanjing Jiancheng Bioengineering Institute) at room temperature for 1 min, followed by dehydration in a gradient of ethanol and clearing in xylene I and xylene II (cat. no. GD-RY1215-12; Shanghai Guduo Science and Technology Co., Ltd., Shanghai, China) twice for 1 min. Stained sections were observed and digital images were captured with a light microscope (magnification, ×400; Zeiss GmbH, Jena, Germany).

PASM staining

Paraffin sections of each group were dewaxed in water, oxidized with a 0.5% periodic acid (cat. no. SBJ-0404; Nanjing SenBeiJia Biological Technology Co., Ltd, Nanjing, China) for 30 min, and washed under tap and distilled water. Following oxidization with 10% chromic acid (cat. no. 06; BaSiFu Co., Ltd, Nanjing, China) for 20 min, the sections were rinsed with distilled water, fixed in a 1% sodium sulfite solution (cat. no. SBJ-0628; Nanjing SenBeiJia Biological Technology Co., Ltd.) for 30 sec, washed with distilled water, and immersed under a hexamine-silver solution (cat. no. SBJ-0600; Nanjing SenBeiJia Biological Technology Co., Ltd.) for 30 min. The sections were left to stand at a temperature of 65°C for a total of 4 min in a water bath, following which they were removed and washed with distilled water and measured under a microscope until the black precipitate appeared in the glomerular capillary basement membrane. Following the appearance of the black precipitate, the sections were colored using a 0.1% auric chloride solution (cat. no. G810493-5 g; Shanghai Chaoyan Biotechnology Co., Ltd., Shanghai, China) for 30 sec, washed under running water for 10 min, and stained with hematoxylin (cat. no. D006; Nanjing Jiancheng Bioengineering Institute) and 0.5% eosin for a total of 3 min. Upon completing a conventional dehydration cycle, the sections were cleared, sealed and observed, and digital images were captured with a light microscope (magnification, ×400; Zeiss GmbH).

Immunohistochemical staining

Paraffin sections were routinely dewaxed into solution, blocked with 3% H2O2 methanol endogenous peroxidases (cat. no. AR1022; Wuhan Boster Biological Technology, Ltd., Wuhan, China), incubated with rabbit anti-rat CD68 monoclonal antibody (cat. no. ab125212; 1:50; Abcam) at 4°C overnight, subsequently incubated with goat anti-rabbit secondary antibodies (cat. no. ab6721; 1:200; Abcam) at room temperature for 30 min, stained with DAB (cat. no. AR1022; Wuhan Boster Biological Technology, Ltd.), counterstained with hematoxylin (cat. no. D006; Nanjing Jiancheng Bioengineering Institute), and dried. Stained sections were observed and digital images were captured with a light microscope (magnification, ×200; Zeiss GmbH).

Cell culture

HK-2 cells and a human monocyte cell line (THP-1) were obtained from the American Type Culture Collection (Manassas, VA, USA). HK-2 and THP-1 cells were cultured at 37°C in RPMI 1640 medium (Lonza Group, Ltd., Basel, Switzerland) containing 10% fetal calf serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 2 mM L-glutamine solution, 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA), in a cell culture incubator with 95% air and 5% CO2 for 24 h. All experiments were performed in serum-free RPMI 1640 medium containing 0.2% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA). Since palmitic acid (PA; Sigma-Aldrich; Merck KGaA) is a major constituent of FFA, it was used to treat the cells and simulate a high fat environment in the in vitro study (22,23).

Transwell filter migration assay and MCP-1 concentration detection

HK-2 cells were seeded in a 6-well plate at a density of 5×105 cells/well and treated with the experimental medium in the absence or presence of 0.32 mM PA or 10 ng/ml Rap plus PA. After a 24-h incubation at 37°C, culture supernatants were harvested via centrifugation at 1,000 × g at 37°C for 10 min. Microporous membrane (pore size, 8 µm) Transwell inserts (Merck KGaA) were used for the chemotaxis assay. THP-1 cells (5×104) in 200 µl serum-free RPMI were added to the upper chamber, with 500 µl of the above culture supernatants in the lower chamber (HK-2 cells were treated with media containing 0.32 mM PA, 10 ng/ml Rap, or PA plus Rap; media without HK-2 cell treatment were used for a control experiment). THP-1 cells were allowed to migrate during a 2-h incubation at 37°C with 5% CO2, and then the inserts were fixed with methanol (cat. no. N168; Shanghai Chaoyan Biotechnology Co., Ltd.) for 20 min at room temperature and stained with 0.1% crystal violet for 10 min at room temperature (cat. no. C0775; Sigma-Aldrich; Merck KGaA). The non-migratory cells were removed prior to mounting of the membrane, and the number of migratory cells was observed under a light microscope (magnification, ×200; Zeiss GmbH). MCP-1 in the supernatant was detected with an MCP-1 ELISA kit (cat. no. ab179886; Abcam), according to the manufacturer's protocol.

Small interfering (si)RNA transfection

HK-2 cells were cultured in six-well plates at a density of 5×105 cells/well and transiently transfected with mTOR siRNA (sense, 5′-CCACCCGAAUUGGCAGAUUTT-3′; antisense, 5′-AAUCUGCCAAUUCGGGUGGTT-3′), FOXO1 siRNA (sense, 5′-GGAGGUAUGAGUCAGUAUATT-3′; antisense, 5′-UAUACUGACUCAUACCUCCTT-3′) or empty vector siRNA (cat. nos. sc-35409 and sc-35382; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer's protocol. Briefly, 1 µg siRNA was mixed with 6 µl Lipofectamine® 2000 and applied to the cells. The transfected cells were incubated at room temperature for 30 min and then seeded (5×105 cells) in a 6-well plate containing 900 µl Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc.). After a 24-h treatment with the experimental medium, the cells were harvested and examined by western blotting.

Protein extraction and western blot analysis

Cellular protein was extracted using a protein extraction kit (cat. no. W034; Nanjing SenBeiJia Biological Technology, Co., Ltd.) and total protein was quantified via a protein assay kit (bicinchoninic acid; cat. no. A045-4; Nanjing SenBeiJia Biological Technology Co., Ltd.). Total protein (30–80 µg) was subjected to electrophoresis on 6–15% SDS polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride membranes, which were blocked for 1 h at room temperature with 5% BSA in Tris-buffered saline containing 0.05% Tween-20 (TBST). Subsequently, blots were washed and incubated overnight at 4°C in TBST containing 5% bovine serum albumin with antibodies (1:1,000 dilution) against interleukin (IL)-1β (cat. no. ab9722; 1:1,000; Abcam), MCP-1 (cat. no. ab25124; 1:1,000; Abcam), p-FOXO1 (cat. no. ab131339; 1:1,000; Abcam), p-mTOR (cat. no. ab137133; 1:1,000; Abcam) and β-actin (cat. no. ab8227; 1:2,000; Abcam). Membranes were washed three times with TBST, incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. ab6721; 1:5,000; Abcam) for 1 h at room temperature and then washed three times again with TBST. Finally, detection procedures were performed using an enhanced chemiluminescence (ECL) Advance Western Blotting Detection kit and autoradiography was performed on Hyperfilm ECL (both GE Healthcare, Chicago, IL, USA). For quantitative analysis, bands were detected and evaluated by densitometry with LabWorks software 7.0 (UVP, LLC, Upland, CA, USA) and normalized to β-actin.

Statistical analysis

Statistical analyses were conducted using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). The χ2 test was applied to assess the distribution of sex between participants with obesity and the controls. Normally distributed variables were analyzed with one-way analysis of variance (ANOVA) or Student's t-test. For variables with non-normal distribution, Kruskal-Wallis' test was used. Tukey's and Dunn's post hoc tests were used after the ANOVA and Kruskal-Wallis' test, respectively. Pearson's correlation analyses were used for continuous variables, and Spearman's correlation analyses were used for ranked variables. P<0.05 was considered to indicate a statistically significant difference.

Results

Baseline characteristics of obese and non-obese subjects

Table I presents the anthropometric and biochemical characteristics of the obese and non-obese subjects. Compared with non-obese controls, the participants with obesity had increased BMI, SBP and DBP, elevated TC, TG and FFA, as well as a larger WC. The Cr, UACR and u-NGAL were increased in the participants with obesity, while there was no significant difference in the BUN between the two groups (Table I). In addition, the participants with obesity had higher concentrations of u-MCP-1 (Table I), and the level of u-MCP-1 was positively correlated with FFA (r=0.507, P=0.002), UACR (r=0.550, P=0.001) and u-NGAL (r=0.448, P=0.007) in the participants with obesity (Fig. 1).

Figure 1. Pearson's correlation between u-MCP-1 levels and FFA, UACR and u-NGAL in patients with obesity. u-MCP-1, urinary monocyte chemoattractant protein-1; FFA, free fatty acid; UACR, urinary albumin to creatinine ratio; u-NGAL, urinary neutrophil gelatinase-associated lipocalin.

Table I. Baseline characteristics of obese and non-obese subjects.

Table I.

Baseline characteristics of obese and non-obese subjects.

Variables	Non-obese (n=27)	Obese (n=35)	P-value
Sex (male/female)	15/12	19/16	0.921
Age (years)	36±9	38±10	0.355
BMI (kg/m2)	22.06±1.45	30.84±2.15a	<0.001
WC (cm)	77.33±6.76	117.80±5.03a	<0.001
SBP (mmHg)	110±4	123±15a	<0.001
DBP (mmHg)	72±7	82±9a	<0.001
TC (mmol/l)	3.37 (2.89, 4.02)	5.48 (3.25, 6.56)a	<0.001
TG (mmol/l)	1.51±0.47	3.29±1.47a	<0.001
FFA (mmol/l)	0.24±0.05	0.45±0.16a	<0.001
BUN (mmol/l)	4.13 (2.62, 5.43)	4.53 (2.56, 5.58)	0.452
Cr (µmol/l)	67.22±10.59	75.69±14.13a	0.012
UACR (mg/g)	4.67±2.35	45.88±37.15a	<0.001
u-NGAL (ng/ml)	6.65±2.25	22.18±15.14a	<0.001
u-MCP-1 (pg/ml)	196.23±61.54	293.02±100.19a	<0.001

{ label (or @symbol) needed for fn[@id='tfn1-mmr-19-02-1284'] } The χ² test was applied to assess the distribution of sex between participants with obesity and the controls. TC and BUN data are expressed as the median and interquartile range, and statistical significance was determined by Kruskal-Wallis' test. All other data are expressed as the mean ± standard deviation; statistical significance was determined by Student's t-test.

a P<0.05 vs. non-obese group. BMI, body mass index; WC, waist circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; TG, triglyceride; FFA, free fatty acid; BUN, blood urea nitrogen; Cr, creatinine; UACR, urinary albumin to creatinine ratio; u-MCP-1, urinary monocyte chemoattractant protein-1; u-NGAL, urinary neutrophil gelatinase-associated lipocalin.

Biochemical characteristics of the rats

BW and the ratio of kidney weight (KW) to BW (KW/BW) was significantly increased in the HFD group, indicating that an obese rat model was successfully established by HFD feeding. Moreover, the levels of serum TG, TC and FFA were markedly higher in HFD-fed rats compared with normal rats. Rap treatment reduced the KW/BW, although it did not notably affect the BW or serum lipids (Table II).

Table II. Biochemical parameters of the NCD, HFD and HFD+Rap groups at the end of the intervention study (n=5).

Table II.

Biochemical parameters of the NCD, HFD and HFD+Rap groups at the end of the intervention study (n=5).

				P-value
Group	NCD	HFD	HFD+Rap	HFD vs. NCD	HFD+Rap vs. HFD
BW (g)	387.33±8.74	462.67±11.37a	459.67±4.51	0.001	0.693
KW/BW (%)	0.85±0.05	1.50±0.08a	1.22±0.10b	<0.001	0.016
TG (mmol/l)	0.35±0.05	1.81±0.03a	1.75±0.11	<0.001	0.405
TC (mmol/l)	1.60±0.10	10.97±0.75a	11.13±0.90	<0.001	0.818
FFA (mmol/l)	1.02±0.08	1.63±0.06a	1.63±0.08	<0.001	1.000
BUN (mmol/l)	5.23±0.35	10.33±0.61a	7.77±0.25b	<0.001	0.003
Cr (µmol/l)	34.67±1.53	68.33±1.53a	37.67±4.04b	<0.001	<0.001

{ label (or @symbol) needed for fn[@id='tfn3-mmr-19-02-1284'] } Data are expressed as the mean ± standard deviation. Statistical significance was determined by one-way analysis of variance.

a P<0.05 vs. NCD group

b P<0.05 vs. HFD group. BW, body weight; KW/BW, kidney weight/body weight; TG, triglyceride; TC, total cholesterol; FFA, free fatty acid; BUN, blood urea nitrogen; Cr, creatinine; NCD, normal chow diet; HFD, high fat diet; Rap, rapamycin.

Renal function of the rats

The Cr, BUN, 24 h urine protein and u-NGAL in the HFD group was increased compared with the NCD group, indicating impaired renal function in these obese rats. By contrast, all the above effects were alleviated in the HFD+Rap group, suggesting that Rap improved the renal function of HFD-induced obese rats (Table II and Fig. 2).

Figure 2. Levels of 24 h urine protein and u-NGAL levels. (A) Elevated 24 h urine protein and (B) u-NGAL levels in HFD-fed rats was alleviated by Rap treatment. Data are presented as the mean ± standard deviation of three individual experiments; statistical significance was determined by one-way analysis of variance. *P<0.05 vs. NCD group; #P<0.05 vs. HFD group. NCD, normal chow diet; HFD, high fat diet; Rap, rapamycin; u-NGAL, urinary neutrophil gelatinase-associated lipocalin.

Renal morphology and IL-1β expression in the rats

HE staining demonstrated vacuolar degeneration in the renal tubular epithelial cells of the HFD group, and PASM staining highlighted basement membrane thickening in the glomeruli and tubules of HFD-fed rats, compared with the NCD group. These effects were alleviated by Rap treatment (Fig. 3). Immunohistochemical staining showed that there was prominent infiltration of CD68+ cells (macrophages) into the renal interstitial area in the HFD group. The tubulointerstitial infiltration of inflammatory cells was significantly decreased by Rap treatment (Fig. 4A). Furthermore, the protein expression of IL-1β was increased in the kidneys of obese rats, compared with the NCD group, while Rap treatment inhibited the expression of IL-1β (Fig. 4B).

Figure 3. HE staining and PASM staining in rat kidney sections (magnification, ×400). HE staining showed significant vacuolar degeneration in renal tubular epithelial cells of the HFD group (the black arrow), and PASM staining showed basement membrane thickening in the glomeruli and tubules of the HFD group. These pathological alterations were reduced by Rap treatment. HE, hematoxylin-eosin; PASM, periodic acid-silver methenamine; NCD, normal chow diet; HFD, high fat diet; Rap, rapamycin.

Figure 4. CD68 and IL-1β staining in rat renal sections. (A) Immunohistochemical staining for CD68 (magnification, ×200). The HFD induced prominent infiltration of CD68+ cells into the renal interstitial area, and Rap decreased the inflammatory cell infiltration. (B) The effects of Rap on the protein expression of IL-1β. Protein expression was examined by western blotting, normalized by comparison with β-actin and expressed as a percentage of control. Data are presented as the mean ± standard deviation of three individual experiments; statistical significance was determined by one-way analysis of variance. *P<0.05 vs. NCD group; #P<0.05 vs. HFD group. NCD, normal chow diet; HFD, high fat diet; Rap, rapamycin; IL-1β, interleukin-1β.

THP-1 monocyte migration

To assess whether PA and Rap were capable of affecting the migration of monocytes, supernatants from HK-2 cells treated with PA, PA plus Rap or Rap alone were tested for their ability to induce monocyte migration using a Transwell migration assay. The supernatants from HK-2 cells treated with PA stimulated THP-1 monocyte migration, and compared with PA, the PA+Rap group stimulated less monocyte migration (Fig. 5A). However, media containing PA, Rap or PA plus Rap that had not been exposed to HK-2 cells had no effect on THP-1 monocyte migration (Fig. 5B), suggesting that PA may mediate monocyte migration by stimulating the secretion of a certain type of chemokine from HK-2 cells, which is potentially inhibited by Rap.

Figure 5. PA stimulates THP-1 monocyte migration. (A) HK-2 cells were treated with experimental medium (Ctr), and medium with 0.32 mM PA, 10 ng/ml Rap, or PA and 10 Rap. After a 24-h incubation, culture supernatants were harvested and used for the chemotaxis assay. The histogram represents the mean ± standard deviation of the THP-1 monocyte counts from three experiments, expressed as a percentage of the Ctr; statistical significance was determined by one-way analysis of variance. *P<0.05 vs. Ctr; #P<0.05 vs. PA. (B) The medium (Ctr) and media containing 0.32 mM PA, 10 ng/ml Rap or PA plus Rap without treating with HK-2 cells were used for a chemotaxis control assay. The media alone did not induce migration. Ctr, control; PA, palmitic acid; Rap, rapamycin.

Protein expression levels of MCP-1, p-FOXO1 and p-mTOR

To test the above hypothesis, the present study detected the expression of MCP-1 in the kidneys of HFD-induced obese rats and in HK-2 cells. The results demonstrated that the protein expression of MCP-1 was significantly increased in the kidneys of obese rats, while Rap treatment inhibited the expression of MCP-1 (Fig. 6A). In addition, PA induced the secretion of MCP-1 from the HK-2 cells, and this was inhibited by Rap, which was consistent with the induction of chemotaxis by the supernatants (Fig. 6B).

Figure 6. MCP-1, p-FOXO1 and p-mTOR expression in the kidneys of rats. (A) Protein expression was examined by western blotting, normalized by comparison with β-actin and expressed as a percentage of control. *P<0.05 vs. NCD group; #P<0.05 vs. HFD group. (B) The release of MCP-1 protein from HK-2 cells. MCP-1 protein content in the supernatant was detected with an ELISA kit. *P<0.05 vs. Ctr; #P<0.05 vs. PA. (C) The protein expression of p-FOXO1 and p-mTOR in HK-2 cells. Protein expression was examined by western blotting. *P<0.05 vs. Ctr; #P<0.05 vs. PA. Data are presented as the mean ± standard deviation of three individual experiments; statistical significance was determined by one-way analysis of variance. NCD, normal chow diet; HFD, high fat diet; Rap, rapamycin; MCP-1, monocyte chemoattractant protein-1; p-FOXO1, phosphorylated forkhead boxO1; p-mTOR, phosphorylated mammalian target of rapamycin; Ctr, control; PA, palmitic acid.

To investigate the potential mechanisms underlying this phenomenon, the protein expression of p-FOXO1 and p-mTOR was assessed in vivo and in vitro. It was found that p-FOXO1 and p-mTOR were upregulated at the protein level (Fig. 6A) in the kidneys of obese rats, while Rap treatment inhibited this change. The in vitro study showed that PA elevated the protein expression of p-FOXO1 and p-mTOR in HK-2 cells, which was inhibited by Rap. Compared with the control, Rap alone had no effect on the expression of these two molecules in HK-2 cells (Fig. 6C). mTOR siRNA or FOXO1 siRNA-transfected HK-2 cells were used to further confirm the above results. The data indicated that mTOR siRNA transfection downregulated the protein expression level of p-FOXO1 (Fig. 7A) and reduced the release of MCP-1 (Fig. 7B) in PA-treated HK-2 cells. Additionally, FOXO1 siRNA transfection increased the secretion of MCP-1 by the HK-2 cells, although not in the PA-treated HK-2 cells (Fig. 7B). Reduced protein expression of mTOR was also observed in the FOXO1 siRNA-transfected HK-2 cells with PA treatment (Fig. 7A).

Figure 7. MCP-1, p-FOXO1 and p-mTOR expression in HK-2 cells transfected with mTOR siRNA or FOXO1 siRNA. (A) The protein expression of p-FOXO1 and p-mTOR in HK-2 cells transfected with mTOR siRNA or FOXO1 siRNA. Protein expression was examined by western blotting. *P<0.05 vs. Ctr; #P<0.05 vs. PA. (B) The release of MCP-1 from HK-2 cells transfected with mTOR siRNA or FOXO1 siRNA. MCP-1 protein content in the supernatant was detected with an ELISA kit. *P<0.05 vs. Ctr; #P<0.05 vs. PA; ΔP<0.05 vs. siFOXO1. Data are presented as the mean ± standard deviation of three individual experiments; statistical significance was determined by one-way analysis of variance. p-FOXO1, phosphorylated forkhead boxO1; p-mTOR, phosphorylated mammalian target of rapamycin; siRNA, small interfering RNA; Ctr, control; PA, palmitic acid; simTOR, siRNA targeting mammalian target of rapamycin; siFOXO1, siRNA targeting forkhead boxO1; HK-2, human renal tubular epithelial cell line; MCP-1, monocyte chemoattractant protein-1.

Discussion

Obesity, a chronic low-grade inflammatory condition, is associated with the development of numerous comorbidities, including renal disease. As more is understood about obesity, the perturbation of renal physiology in the context of obesity has become more apparent (24). In the present clinical study, dyslipidemia was observed in the participants with obesity, and these participants had higher Cr, UACR and u-NGAL, although not BUN, indicating that they had poorer renal function compared with those who were not obese, though not to a severe degree. In addition, the high level of u-MCP-1 was positively correlated with UACR and u-NGAL in the participants with obesity, suggesting that obesity-induced inflammatory responses may contribute to renal injury and dysfunction.

As it would be difficult to obtain kidney samples from the patients with simple obesity, an obese rat model was used to further investigate renal physiology and pathology. It was demonstrated that a HFD provoked overt obesity that was characterized by increased body weight, kidney weight gain and dyslipidemia. Early renal damage was also observed in the HFD-induced obese rats, as evidenced by elevated serum Cr and BUN levels, increased secretion of u-NGAL, proteinuria, vacuolar degeneration in the renal tubules, and glomerular and tubular basement membrane thickening. Additionally, infiltration of CD68+ cells (macrophages) into the renal interstitial area and increased production of inflammatory factors (IL-1β) were found in the HFD-induced obese rats. Further in vitro study showed that supernatants from HK-2 cells treated with PA induced THP-1 monocyte migration. The above results indicated that the renal tubular cells may have been stimulated to release a particular type of chemokine. To explore this hypothesis, the expression of MCP-1 was assessed in vivo and in vitro, since MCP-1 serves an important role in renal inflammatory disease (25,26). The data illustrated that MCP-1 expression was significantly increased in the kidneys of HFD-induced obese rats and in the PA-treated HK-2 cells, which further confirmed the notable correlation between FFA and u-MCP-1 in the participants with obesity. Next, mTOR-FOXO1 signaling pathway protein expression was detected to gain an insight into the mechanism of MCP-1 secretion. The results demonstrated that the expression of p-FOXO1 was elevated in the kidneys of HFD-induced obese rats and in PA-treated HK-2 cells, suggesting that the increased p-FOXO1 had dissociated from the MCP-1 gene promoter and translocated from the nucleus to the cytosol, enabling MCP-1 to be expressed. In addition, the expression of p-mTOR was upregulated in the kidneys of HFD-induced obese rats and in PA-treated HK-2 cells. However, Rap inhibited the expression of MCP-1, p-FOXO1 and p-mTOR, and reduced the inflammatory cell infiltration and IL-1β release in the renal interstitium. Further data revealed that mTOR siRNA transfection downregulated the protein expression level of p-FOXO1 and reduced the release of MCP-1 from PA-treated HK-2 cells. These results suggested that the activated mTOR may have exerted adverse effects in obesity-associated renal tubulointerstitial inflammation. Furthermore, 8 weeks of Rap treatment lowered the Cr and BUN levels, reduced the secretion of urine protein and u-NGAL, improved vacuolar degeneration in the renal tubules and alleviated the glomerular and tubular basement membrane thickening in obese rats, suggesting that inhibition of mTOR may have exerted an anti-inflammatory effect to prevent obesity-associated renal tubulointerstitial inflammation. In the in vitro study, it was also identified that FOXO1 siRNA transfection increased MCP-1 release, indicating that FOXO1 silencing may have reduced MCP-1 promoter inhibition, and MCP-1 was consequently synthesized and secreted. Notably, FOXO1 siRNA transfection reduced the protein expression of p-mTOR in PA-treated HK-2 cells, which may explain the finding that no more MCP-1 was expressed in the PA+FOXO1 siRNA group compared with the PA group. Recently, certain reports have suggested a role for FOXO1 in renal protection (27,28). However, further investigation is required to explore whether FOXO1 is beneficial or detrimental in kidney diseases.

There are a number of limitations to the present study. First, the sample size of this hospital-based cross-sectional study was small, and larger samples are required in the future. Second, the obese animal model does not fully reflect the clinicopathological alterations in participants with obesity, and renal biopsy is necessary to investigate the pathological changes in the kidneys of patients with obesity. Third, other pro-inflammatory factors that have been associated with lower kidney function, such as TNF-α, were not evaluated in the present study (29).

In conclusion, the present findings provided novel evidence, to the best of our knowledge, that activated mTOR induced FOXO1 phosphorylation, which mediated renal MCP-1 release, caused tubulointerstitial inflammation and ultimately lead to renal pathological changes and dysfunction. The inhibition of mTOR may serve a renoprotective role during the progression of obesity-associated renal tubulointerstitial inflammation.

Acknowledgements

Not applicable.

Funding

This work was supported by the National Natural Science Youth Foundation of China (grant no. 81700632 to HS), the Natural Science Youth Foundation of Jiangsu Province (grant no. BK20170366 to HS), and the China Scholarship Council (CSC) (no. 201406090317).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

HS and BS designed the research; HS performed the research, analyzed the data and wrote the paper. XS, JH, MG and BS analyzed the data and revised the paper. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The clinical study protocol was approved by the Institutional Review Board of The First Affiliated Hospital of Soochow University and written informed consent was obtained from each subject. All human rights were observed in keeping with the Helsinki Declaration of 1975, as revised in 2008. The animal study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki, the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Review Board of The First Affiliated Hospital of Soochow University.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References