PPARγ and PGC-1α activators protect against diabetic nephropathy by suppressing the inammation and NF-κB activation

Background: Inammation played critical roles in the progression of various kidney diseases and leaded to irreversible kidney brosis. Peroxisome proliferator-activated receptor gamma (PPARγ) and its coactivator PPARγ coactivator-1 alpha (PGC-1α) negatively regulated mitochondrial biogenesis, cellular energy metabolism, and inammation. But the cooperative molecular mechanism of them in kidney remained unclear. The aim of present study was to investigate this issue. Methods: Human proximal tubular HK-2 cell line was stimulated by inammatory factors, and the expression of PPARγ and its coregulators were determined via reverse transcription-quantitative polymerase chain reaction and western blotting, and DNA binding capacity was measured by EMSA. Furthermore, db/db mice were used to establish a diabetic nephropathy model and administrated with PPARγ and PGC-1α activator. Kidney injury was evaluated microscopically, and inammatory mechanism was assessed by western blotting. Results: Our results revealed that either TNF-α or IL-1b could signicantly decreased PPARγ and PGC-1 expression in vitro. Cytokines also obviously inhibited PPARγ DNA binding activities. Meanwhile, we detected rapid activation of NF-κB pathway under the same experimental conditions. PPARγ and PGC-1α activators effectively protect against diabetic nephropathy and suppress NF-κB expression in db/db mice. Conclusions: PPARγ and its coactivator PGC-1α actively participated in the protection against renal inammation through regulating NF-κB pathway, which highlighted a potential therapeutic target for renal diseases. were separated into three groups (n=6/group) as follows: db/m (C group); db/db (D group) and db/db mice administered 20 mg/kg/day rosiglitazone (R group) or 15 mg/kg/day ZLN005 (Z group) by gavage for 8 weeks. Mice were housed in a specic pathogen-free room at a constant temperature of 22±2˚C and a constant humidity of 50±5%, normal air CO2 and a 12-h light/dark cycle and kept with free access to chow and water. Mice were sacriced after the treatment for 8 weeks. Surgeries were performed under general anesthesia with isourane. All procedures were performed in accordance with the guidelines established for and Laboratory and approval our and Experiments were carried out in accordance guidelines the relevant guidelines and regulation. NF-κB response 5’biotin-AGT-TGA-GGG-GAC-TTT-CCC-AGG-C-3’,3’biotin-TCA-ACT-CCC-CTG-AAA-GGG-TCC-G-5’;mutant-NF-κB response element, 5’biotin-AGT-TGA-GGC-GAC-TTT-CCC-AGG-C-3’, 3’biotin-TCA-ACT-CCG-CTG-AAA-GGG-TCC-G-5’.


Introduction
In ammation is one of the most important factors in the genesis of various complications in renal diseases and leads to renal injury and renal insu ciency. Pro-in ammatory cytokines like tumor necrosis factor-α (TNF-α) and interlukin-1β (IL-1β) play a major role in the onset of acute and chronic in ammatory response in kidney through activating NF-κB pathway, and further induce a wide array of genes encoding pro-in ammatory cytokines, adhesion molecules, chemokines in human and experimental models of kidney diseases (1,2). Our previous studies have described the enrichment in in ammation-related pathways of differential RNAs between diabetic nephropathy and normal control, which indicated the importance of pro-in ammatory mechanisms in diabetic nephropathy (3).
Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear transcription factors and are proved to play key roles in the regulation of lipid metabolism, in ammation, cellular growth and differentiation. Among the various subtypes of PPARs, PPARγ is the best characterized receptor in humans (4) and is widely expressed in the nuclei of mesangial and epithelial cells in glomeruli, vasculatures, proximal and distal tubules, the loop of Henle, and medullary collecting ducts (5). PPARγ also has been veri ed to exert reno-protective effects through an anti-in ammatory mechanism in glomerulosclerosis, glomerulonephritis, microvasculature nephropathy and interstitial in ammation (6-8).
PPARγ regulate the expression of genes through a complex set of mechanisms, and corepressors inhibit and coactivators stimulate PPARγ activity by binding to the PPARγ and changing its conformation without directly binding to the DNA (9,10). Previous researches reported PPARs and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) regulate mitochondrial biogenesis, cellular energy production, thermogenesis, and lipid metabolism (11). We also demonstrated the renoprotective effects against diabetic nephropathy of PPARγ and its coactivator, PGC-1α, through metabolic and antioxidative mechanisms (12).
Investigations have veri ed that expression levels of coregulators are crucial for nuclear receptormediated transcription and part of them have been demonstrated to be targeted for diverse intracellular signaling pathways and post-translational modi cations (13)(14)(15)(16). Moreover, recent studies suggest some coactivators are regulated together with nuclear receptors (such as liver X receptor, retinoid X receptor )in the in ammatory response induced by TNF-α and IL-1β in the heart, liver, brain and adipose tissue (17)(18). But the molecular mechanism in anti-in ammation of PPARγ and PGC-1α in kidney remain to be fully understood.
In the present study, we investigated the expression changes of PPARγ as well as it coactivators, including steroid receptor coactivators (SRCs) and PGC-1α under treatment with TNF-α and IL-1β in vitro, as well as NF-κB pathway activities to primarily explore the mechanism that underlies these changes.
And we further veri ed the effects of activating PPARγ and PGC-1α on diabetic nephropathy and the mechanisms related to NF-κB.
Cell culture HK-2 (the immortalized human proximaltubular cell line, CRL-2190) were purchased from AmericanType Culture Collection (Rockville, MD) and maintained in keratinocyte serum-free media mentioned above. The cells were cultured in a 37°C incubator with 5% CO 2 and subcultured at 80% con uence using 0.05% trypsin-0.02% EDTA (Invitrogen).

Animal experiments
A total of 18 male db/db diabetic mice weighing 32-34 g with a C57BL/KsJ (BKS.Cg-Dock7m+/+Leprdb/Nju) background and 6 male non-diabetic littermate control db/m mice weighing 16-18g (6 weeks old) were obtained from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). They were bred in the laboratory animal center at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China) as previously described (12). Mice were separated into three groups (n=6/group) as follows: db/m (C group); db/db (D group) and db/db mice administered 20 mg/kg/day rosiglitazone (R group) or 15 mg/kg/day ZLN005 (Z group) by gavage for 8 weeks. Mice were housed in a speci c pathogen-free room at a constant temperature of 22±2˚C and a constant humidity of 50±5%, normal air CO2 and a 12-h light/dark cycle and kept with free access to chow and water. Mice were sacri ced after the treatment for 8 weeks. Surgeries were performed under general anesthesia with iso urane. All procedures were performed in accordance with the guidelines established by National Research Council Guide for the Care and Use of Laboratory Animals and approval of our Institute Animal Care and Use Committee (IACUC). Experiments were carried out in accordance with ARRIVE guidelines (https://arriveguidelines.org), the relevant guidelines and regulation.
RNA isolation and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) The total RNA from renal cortical tissues was extracted by using TRIzol (Applied Biosystems, Waltham, MA, USA). The RNA concentration was measured by an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The rst strand of cDNA synthesis was carried out by using a

Western blot analysis
Nuclear and cytosolic fractions of HK-2 cells were separated using NE-PER™ Nuclear and Cytoplasmic Extraction reagents (Thermo Fisher Scienti c, Inc., Waltham, MA, USA) and renal tissues were lysed in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) containing protease inhibitor cocktail (Bimake, Houston, TX, USA) to extract the total protein. The concentration of proteins was determined by Bicinchoninic Acid (BCA) method using BCA Protein Assay kit (Shanghai Epizyme Biotechnology, China). Western blotting was performed as previously described (19). The primary antibodies were obtained from the following sources: anti-PPARγ (Santa Cruz Biotechnology), anti-NLRP3 (Novus Biologicals) , anti-PGC-1α (EMD Millipore), anti-Nephrin and anti-NF-κB (Abcam) . Anti-LaminB and anti-β-actin (Abcam) were used as the loading controls for nuclear and total protein expression, respectively.

MCP-1 assays
Culture supernatants was collected from 6-well plates and concentration of MCP-1 measured by enzyme linked immunosorbent assay (ELISA)using protocols supplied by the manufacturer (R&D Systems, Abingdon, UK) and normalized to cell protein concentrations.

Electrophoretic mobility shift assay (EMSA)
Procedures were performed following standard steps (20). Nuclear extracts containing 10μg protein were added to DNA-binding reaction buffer. Resultant nucleoprotein complexes were analyzed by loading samples on a 6% native polyacrylamide gel in 0.5×TBE buffer, dried, and visualized by autoradiography. In each set of experiments, a self-competition was performed by adding excess of the same unlabeled oligonucleotide probe or a corresponding mutant oligonucleotide probe to the binding reaction at the preincubation step. The following oligonucleotides were used: peroxisome proliferator responseelement Biochemical analysis of serum and urine samples Protein concentration in urine was detected using BCA Protein Assay kit (Shanghai Epizyme Biotechnology, China), and glucose concentration in serum was detected by Glucose LiquiColor® test kit (EFK Diagnostics, Inc., Boerne, TX, USA). After 8 weeks of treatment, urine was collected over 24 h in metabolic cages to measure and calculate the urinary protein excretion (UAE). Blood from the caudal vein was collected for monitoring serum glucose.

Kidney histopathology
Kidney histopathology was performed as previously described (12). Kidneys removed from euthanized mice and were immediately cut in half, xed in 10% formaldehyde in 0.1mol/L PBS (pH 7.2) at 4˚C for 24 h, then embedded in para n and sectioned at 4 µm. The 4-µm-sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol and washed in PBS. Subsequently, for each sample, one section was stained with periodic acid-Schiff (Goodbio Technology CO., LTD, Wuhan, China). Following staining, sections were dehydrated through increasing concentrations of ethanol and xylene.
The general histological alterations in glomerular and tubular structures were evaluated under a light microscope.

Transmission electron microscopy
Renal cortical tissues were xed in 2% glutaraldehyde in phosphate-buffered solution (pH 7.4). Samples were further incubated with 2% osmium tetroxide in phosphatebuffered solution (pH 7.4) for 2 h at 4 °C.
Ultrathin sections were stained with lead citrate and uranyl acetate and viewed on a HT770 transmission electron microscope (Hitachi, Japan) at an accelerating voltage of 80 kV as previously described (21).

Statistical analysis
Data are expressed as mean±SEM. Multiple comparisons were examined for signi cant differences using analysis of variance (ANOVA). Statistical signi cance was set at P<0.05.

Results
TNF-α and IL-1β decrease PPARγ mRNA and protein levels.
As shown in Fig. 1, we rstly investigatedthe dose-response curves and time course of TNF-α or IL-1β on PPARγ expression in HK-2 cells. At the 24h time point, TNF-α induced a dose-dependent decrease in PPARγ mRNA levels, with a nearly 60% decrease at 10ng/ml compared to control group (Paragraph A). IL-1β also decreased PPARγ mRNA level by nearly 50% at the same concentration (Paragraph C).Thereafter, we chose 10ng/ml TNF-α and 10ng/ml IL-1β as the working concentration for subsequent experiments in this study.
Furthermore, at different time points, both TNF-α and IL-1β at 10ng/ml were able to decrease PPARγ mRNA expression as early as 4 h after treatment (Paragraph B and D). We also examined PPARγ protein levels. As shown in Fig. 2, TNF-α or IL-1β at 10ng/ml induced a decrease in PPARγ protein levels at 4h or even earlier at 2h after treatment. Together, these results demonstrated that TNF-α and IL-1β could rapidly suppress PPARγ expression and the decrease is a much sustained cytokine response.
TNF-α and IL-1β decrease the binding of nuclear extracts to PPARγ response elements.
To determine if the decrease of PPARγ induced by TNF-α and IL-1β in uenced binding of nuclear extracts to the peroxisome proliferator response elements(PPRE), we carried out electrophoretic mobility shift assays (EMSA). As shown in Fig. 3, PPARγ binding activities were signi cantly reduced by 10ng/ml IL-1β ( Figure C and D)as early as 2h after treatment and sustained to 24 h. TNF-α ( Figure A and B) also decreased PPARγ binding activities at 2 h after treatment, but recovered gradually since 4h. These data demonstrates that TNF-α and IL-1β could reduce PPARγ binding activity to known cognate response elements for their target genes, especially at early stage of stimulation.
Since TNF-α and IL-1β could greatly decrease expression levels of PPARγ, we examined whether cytokines treatment also have an effect on the expression of coactivators(including SRC-1, SRC-2 and PGC-1). We found the mRNA levels of SRC-1 and SRC-2, which belonged to the p160 family coactivators, were decreased by 35% and 41%within 2 hours after TNF-α (10ng/ml) treatment (Fig. 4A).The expression of PGC-1, another coactivator of PPARγ, was also markedly decreased by 46% as early as 1 hour after TNF-α stimulation (Fig. 4A). Similarly, treatment with IL-1β (10ng/ml) also reduced mRNA level of SRC-1, SRC-2 and PGC-1α by 43%, 58% and 48% separately within 2 hours (Fig. 4B).These results proved that the mRNA levels of the coactivators SRC-1, SRC-2 and PGC-1 could be in uenced together with PPARγ after TNF-α and IL-1β treatment, which may contribute to cytokines-inducedrenal interstitial in ammation.
Finally, we explored the mechanism of the inhibitory effects of TNF-α and IL-1β on PPARγ and its coactivators. We focused on NF-κB pathway which plays critical regulatory roles in in ammation and could be activated by TNF-α and IL-1β directly. Preview investigations have proved that NF-κB pathway has a great relationship with PPARγ and could directly repress PPAR-γ and related genes expression.
We examined the activity of NF-κB after TNF-α and IL-1β stimulation. By EMSA, we found TNF-α or IL-1β could signi cantly promote NF-κB DNA binding activities even after 2 hours treatment, which increased 2.5-fold ( Fig. 5A and 5B)or 2.3-fold over baseline ( Fig. 5C and 5D), respectively. Furthermore, we examined monocyte chemoattractant protein-1 (MCP-1) production after NF-κB pathway activation, which is a downstream target gene of NF-κB pathway and plays important roles in transmigration of in ammatory cells. MCP-1 protein secretion was determined by ELISAas stated in Materials and Methods. We found TNF-α signi cantly increased MCP-1 protein secretion from 15.52 ± 1.05 pg/ml at baseline to 40.47 ± 0.97pg/ml after 24 hours treatment ( Fig. 6 and Table 1). Meanwhile, IL-1β also greatly induced MCP-1 secretion from 19.33 ± 2.33pg/ml at baseline to 160.56 ± 2.8pg/ml even after 8 hours treatment ( Fig. 6 and Table 2).These data veri ed TNF-α and IL-1β effectively induced the activation of NF-κB signaling pathway under same experimental conditions, which mediated down-regulation of PPARγ and coactivators expression. PPARγ and PGC-1α activators protect against diabetic nephropathy and suppress NF-κB expression in db/db mice The body weight and serum glucose levels of the 14 week old db/db mice were signi cantly higher compared with the db/m control group ( Fig. 7A and 7B). The rosiglitazone administration but not ZLN005 reduced serum glucose levels to control levels, and both aginists showed no effects on the body weight of db/db mice after 8 week of administration ( Fig. 7A and 7B). For 14 week old mice, the 24-hour urinary protein levels of the D group was signi cantly increased compared with the C group, while 8 weeks of either rosiglitazone or ZLN005 treatment reduced the 24-hour urinary protein of db/db mice signi cantly (Fig. 7C).
PAS staining revealed obvious mesangical cell proliferation and mesangial matrix expansion in the D group compared with the C group, while either rosiglitazone or ZLN005 treatment aliminated the pathological characteritics markedly and showed only minor differernces compared with the C group ( Fig. 7D). Transmission electron microscopy (TEM) micrographs revealed that podocyte foot process effaced and glomerular basement membrane (GBM) thickened in glomeruli and the density and quantity of mitochondials in tubule cells decreased in db/db mice compared with that in the control mice, and morphological injuries were aliminated after the treatment of rosignitazone or ZLN005 (Fig. 7E and 7F).
The total protein of kidney tissues were extracted and the western blotting results showed a signi cantly reduced expression of nephrin and PGC-1α (Fig. 8A, 8B, 8C, 8D) but signi cantly increased levels of NF-κB and NLRP3 in db/db mice compared with the control mice (Fig. 8E, 8F), and rosignitazone or ZLN005 treatment recovered these changes. These results indicated the renoprotective role of both PPARγ and PGC-1α in diabetic nephropathy with inducing PGC-1α and reducing NF-κB.

Discussion
Extensive studies have con rmed that protective roles of nuclear receptor PPARγ and coactivators in heart, liver, brain and adipose tissue, with the well-de ned mechanism of reguation of in ammatory response, energy metabolism and mitochondrial biogenesis. Our present work revealed that TNF-α or IL-1β administration could induce a rapid and marked decrease in the expression of negative in ammatory protein PPARγ and its coactivators (including SRC-1, SRC-2 and PGC-1) expression in human proximal tubular cells. Activation of NF-κB pathway may be the potential mechanism. Activators of PPARγ and PGC-1α contributed to attenuated the damage in both glomeruli and tubules of diabetic nephropathy in vivo through downregulating the expression of NF-κB and repressing in ammation.
Our data are consistent with previous studies which showed PPARs and coregulators were actively involved in in ammation courses in different human organs. In acute renal failure, Portilla D et al found cisplatin deactivated PPARα by reducing its DNA binding activity and expression of its speci c coactivator PGC-1 (20). Feingold K and colleagues veri ed either administration of LPS in vivo or TNF-α in vitro could obviously decrease the expression of PPARα, -γ and -δ as well as the coactivators (including SRC-1,SRC-2, SRC-3 and PGC-1) in the heart and adipose tissue (22,23). Kim MS group and Rita S group demonstrated similar in ammatory stimulation(including LPS, TNF-α and IL-1β) also decreased the expression of PPARγ, SRC-1, SRC-2 and PGC-1 in the liver cells and uterine smooth muscle cells, but do not in uence expression of SRC-3 and corepressors NCoR and SMRT (24,25).Moreover, Rame JE etal found conditions that induced myocardial steatosis, such as pressure overload, resulted in the decrease of PPARα and PGC-1 levels, which leading to disorders of glycerolipid/free fatty acid cycling (26). In ammation has also been identi ed as a risk factor for cancer and is associated with poor clinical outcome. Investigators found levels of PPARγ, SRC-1 and PGC-1 were obviously lower in breast and colon tumor tissues (27,28), and treatment with anti-tumor drugs could up-regulate SRC-1 and PGC-1 expressions (29,30).All these studies strongly suggested that down-regulation of type II nuclear hormone receptors PPARγ, together with its coactivators, contributed to the progression of in ammatory injuries.
We also explored the molecular mechanism of these effects. NF-κB is a critical transcription factor for maximal expression of many cytokines that are involved in the pathogenesis of in ammatory diseases (31). Moreover, the NF-κB signaling pathway has been represented to be a major avenue for TNF-α or IL-1β inhibition of PPARγ function (32).Under normal conditions, NF-κB is present in the cytoplasm in an inactive state by binding to an inhibitory protein, IκB. In response to activating signals such as TNF-α and IL-1β, IκB is phosphorylated and degraded, which leads to the activation and nuclear translocation of NF-κB (33). Previous studies have certi ed NF-κB activation could inhibit PPARγ function through several approaches. First, NF-κB could cause a direct phosphorylation of serine residues in PPARγ protein, such as Ser112 or Ser82, results in a reduction of its transcriptional activity (34). Second, in acute manner, IκB degradation could cause PPARγ corepressor HDAC3 (Sin3 deacetylase and histone deacetylase) nuclear translocation, and up-regulated binding activities between corepressor complex SMRT/NCoR/HDAC3 with PPARγ, which not only in uences interaction between PPARγ and coactivators, but leads to suppression of the transcriptional activity of PPARγ in the nucleus (34). Moreover, recent studies veri ed NF-κB has direct interaction with PPARγ and its coactivators and resulted in deprivation of the transcriptional coactivators from PPARγ (35,36).In the present study, we also found NF-κB activity was greatly up-regulated along with reduced expression of PPARγ as well as its coactivators and provided new evidences for these inhibitory roles of NF-κB. But the precise mechanism and other possible contributors still need to be explored further.
Since Zhang et al (37) reported ZLN005 as a small molecule inducing PGC-1α expression in mRNA levels in 2013, it has been studied in nerve and cardiovascular tissues and cells on anti-oxidation and mitochondrial damage (38-41). But to out knowledge, there is no report about ZLN005 in kidney disease or anti-in ammatory effects. For the rst time, we employed ZLN005 as the PGC-1α activator in diabetic nephropathy and investigated its mechanism related to in ammatory regulation. These data strongly supported that ZLN005 induced the PGC-1α expression in kidney tissue, which protected against renal in ammatary injury and improved diabetic nephropathy.
TNF-α and IL-1β are systemic and renal local in ammatory mediators. The downregulation by TNF-α and IL-1β of PPARγ and PGC-1α in this work is accompanied by the increase in expression and DNA binding activity of the proin ammatory transcriptional factor NF-κB, which may suggest the molecular mechanism that systemic and renal local in ammation aggravating kidney disease. The renoprotective role of PPARγ and PGC-1α activators through the mechanism involving NF-κB expression and in ammation reduction was veri ed in db/db diabetic nephropathy model mice. In summary, these results highlight that PPARγ together with its coactivator PGC-1α actively participates in the renal in ammation through NF-κB related mechanism. Further exploration of detailed mechanisms may provide novel potential therapeutic targets for renal in ammatory diseases.  Effects of TNF-α and IL-1β on protein expression of PPARγ in HK-2 cells. HK-2 cells were treated for various times in the presence of 10ng/ml TNF-α or IL-1β. (A and C) Nuclear extracts were prepared and Western blot analysis was done using antibodies recognizing PPARγ as described in the Methods section.

Declarations
(B and D) Relative protein levels (% of controls) were reported after normalization with Lamin B, respectively.* P<0.05versus control, △P<0.01versus control.   TNF-α and IL-1β increase the binding of nuclear extracts to NF-κB. HK-2 cells were treated with TNF-α or IL-1βat 10ng/ml as indicated. Nuclear extracts were used for electrophoretic mobility shift assay (EMSA) with oligonucleotides for PPARγ-speci c response elements as described under "Materials and Methods".