Abstract

Background

   Uncoupling protein 1 (UCP1) is predominantly found in brown adipose
   tissue mitochondria, and mediates energy dissipation to generate heat
   rather than ATP via functional mitochondrial uncoupling. However,
   little is known about its expression and function in kidney.

Methods

   We carried out a mRNA microarray analysis in mice kidneys with ischemia
   reperfusion (IR) injury. The most dramatically downregulated gene UCP1
   after IR was identified, and its role in generation of mitochondrial
   reactive oxygen species (ROS) and oxidative stress injury was assessed
   both in vitro and in vivo. Genetic deletion of UCP1 was used to
   investigate the effects of UCP1 on ischemia or cisplatin-indued acute
   kidney injury (AKI) in mice.

Findings

   UCP1 was located in renal tubular epithelial cells in kidney and
   downregulated in a time-dependent manner during renal IR. Deletion of
   UCP1 increased oxidative stress in kidneys and aggravated ischemia or
   cisplatin induced AKI in mice.Viral-based overexpression of UCP1
   reduced mitochondrial ROS generation and apoptosis in hypoxia-treated
   tubular epithelial cells. Furthermore, UCP1 expression was regulated by
   peroxisome proliferator-activator receptor (PPAR) γ in kidneys during
   renal IR. Overexpression of PPAR-γ resembled UCP1-overexpression
   phenotype in vitro. Treatment with PPAR-γ agonist could induce UCP1
   upregulation and provide protective effect against renal IR injury in
   UCP1^+/+mice, but not in UCP1^−/−mice.

Interpretation

   UCP1 protects against AKI likely by suppressing oxidative stress, and
   activation of UCP1 represents a potential therapeutic strategy for AKI.

Fund

   National Natural Science Foundation of China grants, Science and
   Technology Commission of Shanghai.

   Keywords: Acute kidney injury, Uncoupling protein 1, Reactive oxygen
   species, Peroxisome proliferator-activator receptor γ

1. Research in context

   Evidence before this study: Uncoupling protein 1 (UCP1) is
   predominantly found in brown adipose tissue mitochondria, and has a
   central role in adaptive nonshivering thermogenesis in responsible to
   cold exposure via uncoupling ATP synthesis from respiration. Meanwhile,
   UCP1 may reduce reactive oxygen species generation in adipocyte
   mitochondria. However, little is known about its expression and
   function in kidney.

   Added value of this study: In this study, using global gene expression
   profiling approach in a mouse model of renal ischemia reperfusion
   injury, we identify UCP1, which was the most downregulated gene in
   kidney 24 h after renal ischemia reperfusion, as a regulator of kidney
   injury, in particular regulation of reactive oxygen species generation
   and apoptosis. The results demonstrated that UCP1 reduced oxidative
   stress and alleviated ischemia or nephrotoxic drug-induced acute kidney
   injury (AKI)

   Implications of all the available evidence: This study demonstrated the
   crucial role of PPAR-γ/UCP1 signaling pathway in regulating
   ischemia-induced oxidative stress and subsequent kidney injury, which
   is conducive to better understanding the pathogenesis of AKI.
   Activation of UCP1 may be a potential approach for prevention and
   treatment of AKI

2. Introduction

   Acute kidney injury (AKI) is a common clinical complication
   characterised by the rapid loss of renal function, and is often caused
   by renal ischemia reperfusion (IR) and nephrotoxins in the clinics.
   Although supportive treatment is available, AKI is associated with high
   morbidity and mortality, and also contributes to the development of
   chronic kidney diseases [33][1]. Few specific therapies have emerged
   that can prevent or attenuate AKI. It is well known that AKI is a
   common reactive oxygen species (ROS)-related kidney disease [34][2].
   The excess prodution of ROS and a reduction in antioxidant defenses are
   fundamentally implicated in the pathophysiology of AKI. During renal
   ischemia reperfusion injury (IRI), overproduction of ROS occurs early
   in the reperfusion phase and remains for 24 h after reperfusion
   [35][3]. ROS react with proteins, nucleic acids, carbohydrates and
   lipids, contribute to cell apoptosis and necrosis [36][4]. It has been
   demonstrated that the renal proximal tubular cells are particularly
   susceptible to the oxidative stress elicited by ischemia
   reperfusion-induced excess ROS [37][5]. Excess ROS impair the
   mitochondria, trigger inflammation and apoptosis [38][6]. Therefore,
   ROS are one of the critical targets in the management of AKI.

   Mitochondria is the main source of ROS in cells [39][7]. A byproduct of
   mitochondrial respiration is a continual escape of electrons from the
   electron transport chain of the inner membrane which react directly
   with molecular oxygen to form ROS. Uncoupling proteins (UCPs), a family
   of mitochondrial anion carrier proteins expressed in the mitochondrial
   inner membrane, transport protons from inner membrane to the matrix,
   which causes a limited decrease in the inner membrane's potential, and
   thus reduces ROS emission from the electron transport chain, uncoupling
   respiration from ATP production [40][8]. While, when the mitochondrial
   permeability transition occurs, which is associated with the complete
   loss of the membrane potential, ROS burst is observed [41][9]. The
   first described UCP was UCP1 which was predominantly found in brown
   adipose tissue (BAT). UCP1 is critical for thermogenesis in responsible
   to cold exposure via uncoupling ATP synthesis from respiration and
   leading to heat production [42][10]. Moreover, several studies have
   demonstrated that UCP1 may reduce ROS generation by BAT mitochondria
   and thymus mitochondria [43][11], [44][12], [45][13], [46][14].

   In the present study, we carried out a mRNA microarray analysis in mice
   kidneys with IRI, and found UCP1 was the most downregulated gene. UCP1
   was specificly located in the renal tubular epithelial cells in normal
   kidneys. Our in vivo studies together with in vitro experiments in
   which UCP1 was overexpressed or genetically deleted demonstrated the
   critical role of UCP1 in regulating the mitochondrial ROS generation
   and apoptosis associated with ischemia or hypoxic stress. Collectively,
   our data revealed that UCP1 may protect against AKI through inhibiting
   oxidative stress.

3. Materials and methods

3.1. Animals and animal experiments

   Male C57BL/6 J mice were obtained commercially from the Animal Resource
   Center of Fudan University. UCP1^−/− (129S-Ucp1^tm1Kz/J) mice were
   purchased from The Jackson Lab, backcrossed to C57B/6 J mice for 10
   generations, and after repeated intercrossing UCP1^+/+ (wild type) and
   UCP1^−/− strains were obtained and used for experiments. Adult (8- to
   10-week-old) mice used in these experiments were housed in temperature-
   and humidity-controlled cages and fed a standard normal diet with free
   access to rodent food and water. All animal procedures in this study
   were approved by the Institutional Animal Care and Use Committee of
   Fudan University.

   Renal IRI was induced by bilateral renal pedicle clamping for 30 min or
   25 min (mild IRI), as described previously [47][15]. Sham-operated mice
   underwent the same surgical procedures but without occlusion of renal
   pedicle. Cisplatin-induced nephrotoxicity was induced by
   intraperitoneal injection with a single dose of cisplatin at 30 mg/kg,
   or 20 mg/kg (mild cisplatin challenge). To investigate the effects of
   PPAR-γ activation on AKI, we administered pioglitazone (Cayman
   Chemical, Ann Arbor, MI, USA) at a dose of 10 mg/kg/d, or the same
   volume of solvent, by oral gavage for seven consecutive days before IR
   surgery.

3.2. Cell culture and lentivirus transfection

   Mouse renal tubular epithelial cells (mTECs) were purchased from Caltag
   Medsystems (Buckingham, UK), and cultured in Dulbecco's Modified
   Eagle's Medium supplemented with 10% fetal bovine serum, grown in
   incubators at 37 °C and 5% CO[2]. For hypoxia, the cells were
   transferred to a hypoxia incubator with the desired level of hypoxia
   (2% O[2]), and cultivated for 24 h.

   Lentiviral vectors expressing UCP1 or PPAR-γ gene or enhanced green
   fluorescent protein (GFP) were constructed as described previously
   [48][16]. HEK293T cells were used to produce lentiviral particles by
   transfection of lentiviral expression vectors,
   pLV-CMV-STAT3,p80.9,pVSV-G, with pLentivirus-CMV-T2A-GFP, or
   pLentivirus-CMV-UCP1-T2A-GFP, or pLentivirus-CMV-PPAR-γ-T2A -GFP
   plasmids (for overexpression). Seventy-two hours after the
   transfection, the media containing lentiviruses were collected and
   incubated with mTECs for twenty-four hours.

3.3. Microarray hybridization

   Total RNA from kidneys was extracted using Trizol reagent (Invitrogen,
   Carlsbad, USA) according to the manufacturer's instructions. Arraystar
   Mouse LncRNA Microarray V3.0 (KangChen Bio-technology Company,
   Shanghai, China) containing 35 923 LncRNA probes and 24 881 mRNA probes
   was used to detect the global profiling of mouse LncRNAs and mRNAs.
   Sample labeling and array hybridization were conducted according to the
   Agilent One-Color Microarray-Based Gene Expression Analysis protocol
   (Agilent Technology, USA).

3.4. Microarray data analysis

   Agilent Feature Extraction software (version 11.0.1.1) was used to
   extract raw data and analyze acquired array images. GeneSpring GX v12.1
   software package (Agilent Technologies, Santa Clara, CA, USA) was used
   to perform quantile normalization and subsequent data processing.
   Differentially expressed mRNAs with statistical significance in kidneys
   of wild-type mice between the sham group and IR group were identified
   through fold-change filtering (≥ 2.0), multiple hypothesis testing (FDR
   < 0.05), and unpaired t-tests (p < 0.05). Pathway analysis was
   performed to identify differentially regulated biological processes,
   based on KEGG (Kyoto Encyclopedia of Genes and Genomes). LncRNAs data
   was analyzed and further studied in another study. The microarray data
   have been deposited in NCBI Gene Expression Omnibus and are provided
   at: [49]https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131454.
   The accession number is [50]GSE131454.

3.5. Human kidney tissue samples

   We studied three normal controls and three patients with AKI in
   Zhongshan Hospital, Fudan University, between 2014 and 2018. The
   control samples were from normal portions of nephrectomy specimens that
   had been removed for localized renal tumor, and verified by light
   microscopy. Acute tubular necrosis (ATN) was confirmed by renal biopsy
   in these patients with AKI. A ATN patient who had normal renal function
   before operation undergone surgery of ovarian tumor resection,
   accidental hemorrhage and hypotension occurred during the operation,
   serum creatinine was significantly increased 24 h after surgery. Two
   patients with ATN had a history of taking nephrotoxic drugs before
   onset. The renal biopsy tissues were subjected to immunohistochemistry
   staining. The study was approved by the Clinical Research Ethical
   Committee of the Zhongshan Hospital, Fudan University. All patients
   provided written informed consent.

3.6. Assessment of serum creatinine

   Serum creatinine was determined in 100 µl of serum with an automated
   analyzer (Vet test 8008, USA).

3.7. Immunohistochemistry staining

   Immunohistochemistry staining were performed as described previously
   [51][17]. The primary antibody was anti-UCP1 (ab209483, Abcam), and
   horseradish peroxidase-conjugated anti-rabbit IgG was used as secondary
   antibody. Images were visualized under light microscopy.

3.8. Immunofluorescence staining

   Immunofluorescence staining was performed referring to the previously
   published method [52][18]. Anti-UCP1 (ab10983, Abcam) and FITC-labeled
   Lotus tetragonolobus lectin (FL-1321, Vector Laboratories, Burlingame,
   CA) were used as primary antibodies for frozen sections of mouse
   kidney. Secondary Alexa Fluor 555– or Alexa Fluor 488–conjugated
   antibodies against rabbit immunoglobulin (Invitrogen, Carlsbad, CA)
   were used to visualize antigen-antibody complexes. 4′, 6-diamidino-2
   -phenylindole (DAPI) was used for nuclear staining. Images were
   recorded using a confocal microscope (Zeiss LSM 700, Germany).

3.9. ELISA of UCP1

   Concentrations of UCP1 in blood and tissue homogenate were examined by
   commercially available ELISA kit (Wuhan Xinqidi Biological Technology,
   Inc., Wuhan, China), according to the manufacturer's protocol.

3.10. Examination of lipid peroxide

   The level of malondialdehyde (MDA) in mouse renal tissue homogenate was
   tested using commercially available kit (TBARS Assay Kit, Cayman
   Chemical Company, USA), according to the manufacturer's protocol.

3.11. Flow cytometry

   Cell apoptosis was detected by flow cytometric analysis with an Annexin
   V/PI kit (BD Bioscience, USA) according to the manufacturer's
   instructions. Cells with Annexin V (FITC) or propidium iodide (PI)
   staining or not were detected by flow cytometry (Beckman Coulter, CA,
   USA) and analyzed by FlowJo 7.6 software.

3.12. Mitochondrial ros generation assay

   Cell mitochondrial ROS generation was detected using MitoSOX™ Red
   mitochondrial superoxide indicator (Invitrogen) according to the
   manufacturer's instructions. Images were recorded by confocal
   microscopy. Tissue ROS production was detected on frozen kidney
   sections using Dihydroethidium (DHE) staining, as described previously
   [53][19]. Images were captured by confocal microscopy, and quantified
   morphometrically to measure area of fluorescent signal with Image-J.

3.13. Mitochondrial membrane potential assay with JC-1 staining

   Mitochondrial membrane potential (MMP) assay kid (Beyotime, China) was
   used to detect MMP in mTECs according to the manufacturer's
   instructions. In healthy cells with high MMP, JC-1 enters mitochondria
   matrix, forming a polymer (j-aggregates) with intense red fluorescence.
   While, in unhealthy cells with low MMP, JC-1 remains as a monomer,
   shows only green fluorescence. Thus, MMP can be detected by the change
   of fluorescence color and analyzed by a flow cytometer. In brief, cells
   were collected and 500 μL JC-1 staining solution was added, and
   incubated for 20 min. Then cells were resuspended in 500 μL preheated
   JC-1 assay buffer, and analyzed using flow cytometer. Data are
   presented as mean of red/green fluorescence ratio.

3.14. Assessment of cell viability

   Cell viability was determined by cell counting kit-8 (CCK8; Dojindo,
   Kumamoto, Japan) according to manufacturers’ instructions. In brief,
   mTECss were treated with CCK-8 regent (100μL/well) and incubated for
   2 h. The absorbance was measured at 450 nm with a microplate reader,
   and the cell viability was calculated.

3.15. Assessment of the activity of mitochondrial respiratory chain complex
i/iii

   The mitochondrial respiratory chain complex (MRCC) I/III kits
   (Solarbio, Beijing, China) were used to test the activity of MRCC
   I/III, as previously described [54][20]. In brief, 5 × 10^6 mTECs were
   homogenized on ice, centrifuged and the precipitant (mitochondria) was
   suspended, then treated by ultrasonication. The samples were mixed with
   working solution, and the initial absorbance (A1) and final absorbance
   after 2 min (A2) were recorded at 340 nm for MRCC I and at 550 nm for
   MRCC III, respectively. The activity of MRCC I/III was calculated.

3.16. Real-time polymerase chain reaction (PCR)

   Total RNA from mTECs and dissected kidney tissue was extracted using
   Trizol reagent (Invitrogen, Carlsbad, CA), and then reverse-transcribed
   to complementary DNA (PrimeScript RT reagent Kit; TaKaRa, Japan),
   followed by real-time PCR (SYBR Premix Ex TaqTM TaKaRa). The PCR primer
   sequences were presented in Supplemental Table 1. The analysis method
   was as previously described [55][14].

3.17. Western blot

   Western blot was performed as previously described [56][15]. The
   primary antibodies were used as follows: anti-UCP1 (ab209483, Abcam),
   anti-PPAR-γ (sc-7273, Santa Cruz Biotechnology), anti-Caspase 3 (#9662,
   Cell Signaling Technology) and anti-Cleaved Caspase 3 (#9661, Cell
   Signaling Technology), anti-GAPDH (sc-365,062, Santa Cruz
   Biotechnology).

3.18. Statistical analysis

   Statistical analysis was performed using the SPSS Version 16.0
   (Chicago, IL). Data were expressed as a mean ± SEM. The differences
   between two groups were analyzed by two-tailed, unpaired t tests. For
   comparison of means among multiple groups, one-way ANOVA followed by
   Bonferroni posttest was applied. A P value less than 0.05 was
   determined to be significant.

4. Results

4.1. Renal ischemia reperfusion induces downregulation of UCP1

   To systematically identify mRNAs involved in ischemic kidney injury, we
   used mRNA microarray to identify differentially expressed mRNAs during
   renal IR. The results showed that 267 mRNAs were differentially
   expressed after renal IR (fold change ≥2; P < 0.05), among which 192
   were upregulated and 75 were downregulated. An expression volcano map
   of the differentially expressed mRNAs showed that UCP1 was most
   downregulated ([57]Fig. 1A). Next, qRT-PCR was performed to analyze the
   expression of the top 11 downregulated mRNAs (fold change > 4) in the
   mouse kidneys 24 h after renal IR. The expression of most mRNAs was
   consistent with the microarray analysis except in the cases of
   [58]NM_007702 and [59]NM_009605, and [60]NM_009463 (UCP1) was the most
   downregulated mRNA ([61]Fig. 1B). Then, we detected the UCP1 expression
   in multiple organs in wild-type (WT) mice, and found it was specificly
   expressed in kidney, not in heart, liver and lung ([62]Fig. 1C). While
   there was no expression of UCP1 in these organs in UCP1^−/− mice (data
   not shown). UCP1 was present in the cytoplasm of tubular epithelial
   cells in both cortex and medulla of kidney ([63]Fig. 1C). Furthermore,
   we examined the expression profiles of UCP1 mRNA during renal IR and
   found it was decreased in a time-dependent manner, and reached the
   lowest level at 24 h after renal IR ([64]Fig. 1D). In the protein
   level, UCP1 was also downregulated after renal IR in kidneys and in the
   blood ([65]Fig. 1E and Supplemental Fig. 1A,B).

Fig. 1.

   [66]Fig 1
   [67]Open in a new tab

   Renal ischemia reperfusion (IR) induces downregulation of UCP1 in mice.
   (A) A volcano plot showed the differentially expressed mRNAs in IR
   group compared to Sham group. Red and green dots represent upregulated
   and downregulated mRNAs in kidneys 24 h after ischemia reperfusion
   (IR), respectively (fold change ≥2.0 and P-value ≤0.05). Black dots
   represent the genes that were not differentially expressed. UCP1 was
   the most downregulated gene. (B) Quantitative RT-PCR analysis of the
   top 11 downregulated mRNAs in the mouse kidneys 24 h after renal IR.
   (C) Immunohistochemical staining for UCP1 in multiple organs in mice.
   UCP1 was specificly expressed in kidney, including renal cortex and
   medulla (arrow). Scale bar, 100 μm. (D) Quantitative RT-PCR analysis of
   UCP1 mRNA during renal IR. (E) Enzyme-linked immunosorbent assay
   (ELISA) of UCP1 in kidney. Data represent mean ± SEM. n = 4–6. **P <
   0.01 versus Sham group. (F) Representative images of
   immunohistochemical staining for UCP1 in kidneys from patients with
   acute kidney injury (AKI) or normal controls. UCP1 was expressed in
   renal tubular epithelium (arrow). Scale bar, 100 μm. The control
   samples were from normal portions of nephrectomy specimens that had
   been removed for localized renal tumor. Acute tubular necrosis (ATN)
   was confirmed by renal biopsy in the patients with AKI.

   In addition, we detected UCP1 expression in kidneys from patients with
   ATN caused by hemorrhage or nephrotoxic drugs, or normal controls. More
   detailed information about the patients and normal controls is
   available in the Materials and methods. Similarly, UCP1 was widely
   expressed in renal cortex and medulla of normal control kidneys, while,
   in kidneys of ATN patients, UCP1 expression was significantly
   downregulated ([68]Fig. 1F).

4.2. Deletion of UCP1 aggravates acute kidney injury

   To further determine the role of UCP1 in the pathogenesis of AKI, we
   subjected UCP1^−/− mice and WT littermates to mild renal ischemia or a
   mild dose of cisplatin. The results showed that 25-minute clamping of
   bilateral renal pedicle did not cause an increase of serum creatinine
   24 h after reperfusion in WT mice, while a significant increase of
   serum creatinine was found in UCP1^−/− mice ([69]Fig. 2A). Similarly, a
   mild single dose of cisplatin (20 mg/kg) caused a significant increase
   in serum creatinine in UCP1^−/− mice, but not in WT mice ([70]Fig. 2B).
   Next, we used DHE staining to assess the change in production of ROS,
   and found UCP1^−/−mice showed a higher production of ROS in kidney
   tissue 24 h after renal IR compared with WT mice ([71]Fig. 2D).
   Meanwhile, the UCP1^−/− mice had higher level of MDA in kidneys
   compared with WT mice ([72]Fig. 2C). These results indicated that UCP1
   could inhibit oxidative stress and conferred protective effects against
   AKI.

Fig. 2.

   [73]Fig 2
   [74]Open in a new tab

   Deletion of UCP1 aggravates acute kidney injury and increased reactive
   oxygen species generation. (A) Serum creatinine 24 h after renal IR.
   25-min renal ischemia induced a significant increase in serum
   creatinine in UCP1^−/− mice, but not in wild-type (WT) mice. (B) Serum
   creatinine 72 h after cisplatin injection. Treatment with 20 mg/kg
   cisplatin (Cis20) caused a significant increase in serum creatinine in
   UCP1^−/− mice, but not in WT mice. (C) Malondialdehyde (MDA)
   concentration in mice kidneys. (D) Images and quantification of
   dihydroethidium (DHE) staining for detection of reactive oxygen species
   (ROS) generation in mice kidneys. Scale bar, 100 μm. Data represent
   mean ± SEM. n = 6. *P < 0.05, **P < 0.01 versus WT-IR25’ or WT-Cis20’
   group.

4.3. UCP1 overexpression reduces mitochondrial ros generation and apoptosis
in hypoxia-treated tubular epithelial cells

   To further investigate the effects of UCP1 overexpression on apoptosis
   and ROS generation in tubular epithelial cells treated with hypoxia,
   UCP1 was overexpressed in mTECs by transfection with UCP1
   overexpressing lentivirus (UCP1-Lenti). Negative lentivirus (Neg-Lenti)
   was as control. The expression of UCP1 was significantly higher in
   mTECs infected with UCP1-Lenti than in that of Neg-Lenti (Supplemental
   Fig. 2A). We investigated the effects of UCP1 overexpression on
   mitochondria membrane potential (MMP) together with viability in mTECs.
   The results showed that UCP1 overexpression induced a mild decrease in
   MMP, while, did not affect cell viability (Supplemental Fig. 2B, C).
   Meanwhile, we examined the effects of UCP1 overexpression on
   mitochondria respiratory parameters, complex I and III, in mTECs. The
   results revealed that UCP1 overexpression did not affect the activity
   of mitochondria respiratory chain complex I and III (Supplemental Fig.
   2D, E). As expected, overexpression of UCP1 significantly decreased
   mitochondrial ROS generation in mTECs treated with hypoxia, and also
   reduced apoptosis ([75]Fig. 3A,C). Next, we examined the involvement of
   UCP1 overexpression in the expression of apoptosis-related protein
   caspase 3 in vitro. The results showed that the cleaved caspase 3
   expression in mTECs transfected with UCP1-Lenti was significantly
   decreased compared to those transfected with Neg-Lenti in hypoxia
   (Supplemental Fig. 2F). In addition, we investigated the effect of UCP1
   overexpression on mitochondrial membrane potential (MMP) in tubular
   epithelial cells treated with hypoxia. We found that the loss of MMP
   was significant in Hypoxia group. While, the MMP was significantly
   higher in UCP1 overexpression group than Hypoxia group ([76]Fig. 3B).

Fig. 3.

   [77]Fig 3
   [78]Open in a new tab

   UCP1 overexpression suppresses mitochondrial reactive oxygen species
   generation and apoptosis. (A) Images and quantification of
   mitochondrial reactive oxygen species (ROS) generation from
   hypoxia-treated mTEC transfected with UCP1 overexpressing lentivirus
   (UCP1-Hypoxia) or Negative lentivirus (Neg-Hypoxia). Scale bar, 25 μm.
   (B) Mitochondrial membrane potential was examined via JC-1 staining.
   Red inflorescence indicates the healthy mitochondria, and green
   inflorescence indicates the loss of mitochondrial potential. (C) Cell
   apoptosis in hypoxia-treated mTEC transfected with UCP1 overexpressing
   lentivirus or Negative lentivirus. The percentages of apoptotic cells
   were analyzed by Annexin V/PI analysis, and Annexin V positive cells
   were considered as apoptotic cells. Data represent mean ± SEM. **P <
   0.01 versus Hypoxia group.

4.4. PPAR-γ regulates UCP1 expression during renal ischemia reperfusion

   Pathway analysis indicated that genes associated with PPAR signaling
   pathway were most downregulated by renal IR ([79]Fig. 4A). It is well
   documented that the transcription of UCP1 is regulated by several key
   tissue-restricted transcription factors, including peroxisome
   proliferator-activator receptor (PPAR) α, γ, and PPAR-γ co-activator
   (PGC)−1α [80][21], [81][22]. We detected the expression of PPARs in
   kidneys during IR. The results revealed that PPAR-α and PGC-1α mRNAs
   did not alter (Supplemental Fig. 3A,B), however, PPAR-γ mRNA was
   decreased in a time-dependent manner ([82]Fig. 4B), which was
   consistent with the expression of UCP1 ([83]Fig. 1D). Western blot
   analysis showed that both PPAR-γ and UCP1 were downregulated in kidneys
   6 h and 24 h after renal IR (Supplemental Fig. 3C). In vitro, PPAR-γ
   and UCP1 proteins were also decreased in mTECs treated with hypoxia
   ([84]Fig. 4C). We further examined the effect of PPAR-γ overexpression
   on the expression of UCP1 in mTECs. PPAR-γ was overexpressed in mTECs
   by transfection of PPAR-γ overexpressing lentivirus (Supplemental Fig.
   4). As expected, overexpression of PPAR-γ significantly upregulated
   UCP1 expression ([85]Fig. 4D). Collectively, these results suggested
   that UCP1 was regulated by PPAR-γ in kidney.

Fig. 4.

   [86]Fig 4
   [87]Open in a new tab

   PPAR-γ regulates UCP1 expression during renal ischemia reperfusion. (A)
   KEGG pathway enrichment analysis for downregulated mRNAs. Genes
   associated with PPAR signaling pathway were most downregulated 24 h
   after renal ischemia reperfusion (IR). (B) qRT-PCR analysis of PPAR-γ
   mRNA during renal IR in wild-type mice. Data represent mean ± SEM. **P
   < 0.01 versus Sham group. (C) Western blot analysis of UCP1 and PPAR-γ
   in hypoxia-treated mTECs. (D) Western blot analysis of PPAR-γ and UCP1
   in mTEC transfected with PPAR-γ overexpressing lentivirus (PPAR-γ-OE)
   or Negative lentivirus (Neg). PPAR-γ overexpression upregulated UCP1
   expression. *P < 0.05, **P <0.01. KEGG, Kyoto Encyclopedia of Genes and
   Genomes.

4.5. Overexpression of PPAR-γ inhibits hypoxia-induced ros generation and
apoptosis in mTECs

   We then investigated whether overexpression of PPAR-γ resembled
   UCP1-overexpression phenotype in vitro. PPAR-γ was overexpressed in
   mTECs by transfection with PPAR-γ overexpressing lentivirus
   (PPAR-γ-Lenti). Negative lentivirus (Neg-Lenti) was as control.
   Similarly, overexpression of the PPAR-γ significantly decreased
   mitochondrial ROS generation in mTECs treated with hypoxia, and also
   reduced apoptosis ([88]Fig. 5A,B).

Fig. 5.

   [89]Fig 5
   [90]Open in a new tab

   PPAR-γ overexpression inhibites hypoxia-induced ROS generation and
   apoptosis in vitro. (A) Images and quantification of mitochondrial ROS
   generation from hypoxia-treated mTECs transfected with PPAR-γ
   overexpressing lentivirus (PPAR-γ-Hypoxia) or Negative lentivirus
   (Neg-Hypoxia). Scale bar, 25 μm. (B) Cell apoptosis in hypoxia-treated
   mTECs transfected with PPAR-γ overexpressing lentivirus or Negative
   lentivirus. The percentages of apoptotic cells were analyzed by Annexin
   V/PI analysis, and Annexin V positive cells were considered as
   apoptotic cells. n = 4. Data represent mean ± SEM. **P < 0.01 versus
   Hypoxia group.

4.6. PPAR-γ agonist alleviates renal ischemia reperfusion injury via UCP1

   Previous studies demonstrated that chronic treatment of PPAR-γ agonist
   could induce UCP1 upregulation in BAT and white adipose tissue
   [91][23], [92][24]. Then, we investigated the effect of PPAR-γ agonist
   on the expression of UCP1 in kidney. Mice were orally administered with
   pioglitazone (10 mg ⁄ kg ⁄ day), a selective PPAR-γ agonist, or solvent
   for one week. The result revealed that PPAR-γ activation significantly
   upregulated UCP1 mRNA expression in kidney ([93]Fig. 6A). Next, we
   examined whether PPAR-γ activation would provide protective effect
   against renal IRI and whether this effect was mediated by UCP1.
   UCP1^−/− and WT mice were orally administered with pioglitazone or
   solvent for one week, and then subjected to renal IR. The results
   revealed that pioglitazone administration markedly reduced
   concentration of serum creatinine when compared to solvent
   administration in WT mice, but not in UCP1^−/− mice ([94]Fig. 6B).
   Moreover, pioglitazone administration caused a significant decrease in
   MDA in kidneys of WT mice when compared with that of UCP1^−/− mice
   ([95]Fig. 6C). Furthermore, pioglitazone administration upregulated
   UCP1 expression in kidneys in WT mice ([96]Fig. 6D). These data
   indicated that PPAR-γ alleviated renal IRI via upregulation of UCP1.

Fig. 6.

   [97]Fig 6
   [98]Open in a new tab

   PPAR-γ agonist upregulates UCP1 expression and attenuates
   ischemia-induced acute kidney injury and oxidative stress. (A) PPAR-γ
   agonist upregulated UCP1 mRNA expression in kidney. Mice were orally
   administered with PPAR-γ agonist pioglitazone (Piog, 10 mg ⁄ kg ⁄ day),
   or solvent (Solv) for one week. Data represent mean ± SEM. n = 6. **P <
   0.01. (B) Concentration of serum creatinine 24 h after renal ischemia
   reperfusion (IR). UCP1^−/− (UCP1 KO) and wild-type (WT) mice were
   orally administered with PPAR-γ agonist pioglitazone or solvent for one
   week, and then subjected to renal IR. (C) Malondialdehyde (MDA)
   concentration in kidneys 24 h after renal IR in WT and UCP1^−/− mice.
   Data represent mean ± SEM. n = 6. **P <0.01 versus IR group. (D)
   Western blot analysis of UCP1. Data are means from three independent
   experiments. *P < 0.05. (E) Proposed schema of the pathway underlying
   renal ischemia reperfusion-induced acute kidney injury, involving
   downregulation of PPAR-γ and UCP1, promotion of mitochondrial reactive
   oxygen species (ROS) generation and oxidative stress.

5. Discussion

   In this study, using global gene expression profiling approach in a
   mouse model of renal IRI, we identified UCP1 as a regulator of kidney
   injury, in particular regulation of ROS generation and inhibition of
   apoptosis. Renal ischemia induced downregulation of PPAR-γ, which
   inhibited UCP1 expression, and subsequently promoted mitochondrial ROS
   generation, resulting in oxidative stress injury and AKI ([99]Fig. 6E).
   Moreover, deletion of UCP1 increased the generation of ROS, displayed
   greater susceptibility to renal IRI or nephrotoxicity. As such,
   activation of UCP1 represents an approach for new therapeutics to treat
   AKI.

   As mentioned above, UCPs play an important role in the generation of
   mitochondrial ROS. There are five UCPs (UCP-1 to −5) which are somewhat
   different in tissue distributions and functions. As the first
   identified and most studied uncoupling protein, UCP1 function and its
   expression have been highly contentious. Previous evidences suggested
   that UCP1 expression was limited to fat tissues [100][25], [101][26],
   most abundant in the BAT. Here, we found UCP1 was also expressed in
   normal kidney, with widespread expression in the renal tubules
   ([102]Fig. 1C and [103]Fig. 1F), but not in heart, liver and lung.
   Early evidences showed that UCP1 had no effect on ROS generation and
   other markers of oxidative stress [104][27]. However, growing evidences
   have demonstrated that UCP1 activation may reduce mitochondrial ROS
   generation in BAT, beige adipose tissue, thymus and skeletal muscle
   [105][11], [106][12], [107][13], [108][28]. Consistent with these
   results, our data indicated that UCP1 significantly reduced
   mitochondrial ROS generation in hypoxia-treated mTECs and ROS
   generation in ischemic kidneys from UCP1^−/− mice was significantly
   higher than that from WT mice. UCP2 has high similarity with UCP1 in
   sequence, and is widely expressed in several tissues including kidney,
   heart, lung, and spleen [109][29], [110][30], [111][31]). In this
   study, our data indicated that UCP2 was upregulated in kidneys 24 h
   after IR (Supplemental Fig. 5), which was consistent with previous
   studies [112][32]. Upregulation of UCP2 could provide protective
   effects against renal IRI [113][32].

   It is noteworthy that mitochondrial uncoupling induced by UCP1 causes a
   reduction in the oxidative phosphorylation, affecting the ATP
   production which is particularly important for proximal tubule
   epithelial cells to maintain cellular function. Whether mitochondrial
   uncoupling compromise the excretion and reabsorption process of tubular
   epithelia cells are not explored in this study. Recent evidence has
   indicated that the induction of mitochondrial uncoupling not only
   impacts mitochondrial respiration but also regulates multiple cellular
   biological processes, including autophagy, ROS production, protein
   secretion, physical exercise capacity [114][33], [115][34], [116][35],
   [117][36]. Further experiments would be needed to elaborate these and
   improve our knowledge of the cellular consequences of mitochondrial
   uncoupling in resident kidney cells.

   It has been demonstrated that the transcription of UCPs are regulated
   by PPAR-α, γ, and PGC-1α [118][37]. In vivo PPAR-γ activation can
   induce expression of UCP-1, −2, and −3 in BAT [119][21], [120][23],
   [121][38]. In the current study, pathway analysis indicated that genes
   associated with PPAR signaling pathway were most downregulated during
   renal IR ([122]Fig. 4A), and we found that PPAR-γ mRNA was decreased
   consistent with the expression of UCP1 ([123]Fig. 4B and [124]Fig. 1D).
   Given the potential of PPAR-γ for regulation of UCP-1 expression, and
   that PPAR-γ acts as a regulator of energy homeostasis, we assessed the
   effects of PPAR-γ activation on UCP1 expression in kidneys, oxidative
   stress and renal damage induced by renal IR. Interestingly, we
   discovered that mice treated with pioglitazone, a selective PPAR-γ
   agonist, exhibited higher level of UCP1 protein in kidneys, lower MDA
   and attenuated renal injury, compared with the solvent control group
   mice. However, administration of pioglitazone did not attenuate
   oxidative stress and renal injury in UCP1^−/− mice. It suggests that
   PPAR-γ activation may be associated with UCP1-mediated inhibition of
   oxidative stress and subsequent improvement of renal IRI.

   In this study, PPAR-γ agonist (pioglitazone) was administered before
   the onset of renal injury, which is sort of limitation to perform in
   clinic. Nevertheless, in fact, it is possible to identify patients at
   high risk of AKI, such as major surgical procedures, severe infection,
   nephrotoxic drugs administration, especially in critically ill
   patients. Pre-activation of UCP1 may be beneficial for prevention of
   AKI.

   The present study demonstrated the involvement of UCP1 in the
   beneficial response to AKI via inhibiting oxidative stress and
   apoptosis. PPAR-γ agonist provided protective effects against renal IRI
   through activation of UCP1. Further experimental studies and clinical
   trials would be valuable in insight into activation of UCP1 and its
   clinical application.

Author contributions

   P.J. and X.D. designed the study; P.J., T.P., X.W., S.X. and J.H.
   carried out experiments; P.J., X.W, X.D., and T.P. analyzed the data;
   P.J., X.D. wrote the manuscript.

Declaration of Competing Interest

   The authors declare that they have no conflict of interest.

Acknowledgments