Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disorder and a leading cause of genetic end-stage renal disease (Igarashi and Somlo, 2007). It affects one in thousand individuals and is caused by mutations in either
Forkhead box protein O (FOXO) transcription factors, including FOXO1 (FKHR), FOXO3 (FOXO3a, FKHRL1), FOXO4 (AFX), and FOXO6 in mammals (Calnan and Brunet, 2008), are negatively regulated by the phosphatidylinositol 3-kinase (PI3K)/serine-threonine kinase (AKT) signaling pathway. This AKT-mediated phosphorylation of FOXO results in its exportation into the cytoplasm and enhancement of proteasomal degradation (Tzivion
In this study, we aimed to identify novel mechanisms associated with oxidative stress and cystogenesis in ADPKD. We observed that Foxo3a was a key regulator of ROS accumulation in a
This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Seoul National University Hospital (Seoul, Korea) (H-0701-033-195). Informed consent was obtained from all patients. Renal cystic tissues were obtained from the renal cortex surrounding the cysts of patients with ADPKD while undergoing nephrectomy. Non-ADPKD renal tissue were obtained from patients with clear cell renal cell carcinoma (RCC) as a control, and malignant cell infiltration was excluded by histology. All these tissues were also used in our previous study (Woo
Inner medullary collecting duct cells (IMCD cells; CRL-2123™, ATCC, Manassas, VI, USA) were cultured in DMEM/F12 medium (LM002-04, Welgene, Gyeongsan, Korea) containing 10% foetal bovine serum (FBS; 26140-079, Gibco, Waltham, MA, USA), and 1% penicillin-streptomycin (LS 202-02, Welgene). Normal human renal cortical epithelial cells (HRCE; CC-2554, LONZA, Morristown, NJ, USA) were cultured in REGM medium (CC-4190, LONZA) containing 1% penicillin-streptomycin. WT9-7 (CRL-2830™, ATCC), a proximal cortical tubule epithelial cell line isolated from renal cysts from a patient with ADPKD, was cultured in DMEM (LM001-05, Welgene) containing 10% FBS and 1% penicillin-streptomycin in culture dishes coated with type I bovine collagen (#354231, Corning, New York, NY, USA). Human embryonic kidney 293T cells (HEK293T) were cultured in DMEM (LM001-05, Welgene) containing 10% FBS and 1% penicillin-streptomycin in Poly-D-lysine-coated dishes (P7280, Sigma-Aldrich, St. Louis, MO, USA). These cells were grown in a humidified 5% CO2 incubator at 37°C. For hypoxic conditions, cells were incubated in 5% CO2 and 1% O2 balanced with N2 in a hypoxic chamber within an incubator at 37°C.
Total RNA was extracted from cell lines and kidney tissues using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. To assess miRNA expression, first, cDNA was prepared from total RNA (1 μg) using the miScript II RT kit (Qiagen) according to the manufacturer’s protocol. Then, quantitative real-time PCR was performed using miScript Primer Assays (Qiagen) and the miScript SYBR Green PCR Kit (Qiagen) according to the manufacturer’s protocol. RNU6 was used as an endogenous miRNA control.
The mouse and human
miRNA mimics (Ambion) or inhibitors (Dharmacon, Lafayette, LA, USA) of miR-132-3p were reverse transfected into cell lines (at 30 nM) using siPORT NeoFX transfection reagent (Ambion) for 48 h. For the control experiments cells were transfected with a negative control miRNA mimic (mirVana™ miRNA Inhibitor, Negative Control #1, Ambion) or the inhibitor (miRIDIAN microRNA Hairpin Inhibitor Negative Control #1, Dharmacon). Small interfering RNAs (siRNAs) targeting the mouse or human Fox3 gene (Santa Cruz, Dallas, TX, USA) were transfected into cells (at 20 nM) using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions. Subsequent experiments were performed 48 h after transfection.
mRNA was isolated with the NucleoSpin® RNA/Protein kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instructions. For mRNA expression analysis, The isolated RNA (1 μg) was reverse-transcribed to cDNA using M-MLV Reverse Transcriptase (Promega), RNase inhibitor (Promega), oligo-dT (100 nM), and dNTPs (2.5 mM; Promega). Quantitative real-time PCR was performed using qPCRBIO SyGreen Blue Mix (PCR Biosystem, London, UK) and a LightCycler® 96 System (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Mouse β-actin and human 18S rRNA were used as endogenous controls.
Proteins were isolated using the NucleoSpin® RNA/Protein kit (Macherey-Nagel), according to the manufacturer’s protocol. The protein concentration was calculated using the BCA assay and bicinchoninic acid and copper (II) sulphate solutions (Sigma-Aldrich), and equal amounts of protein were separated by 8-12% SDS-PAGE and were electro-transferred to polyvinylidene fluoride (PVDF) membranes (ATTO, Tokyo, Japan). Immunoblots were probed with primary antibodies against the following proteins: FoxO3a (#2497, Cell Signaling, Danvers, MA, USA), HIF-1α (#14179, Cell Signaling), GATM (ab228937, Abcam, Cambridge, MA, USA), ALDH4A1 (ab185208, Abcam), ALDH1L1 (sc-100497, Santa Cruz), ALDH6A1 (sc-271582, Santa Cruz), COX IV (ab33985, Abcam), α-tubulin (#3873, Cell Signaling), and β-actin (A300-491A, Bethyl Laboratories, Montgomery, AL, USA). The primary antibodies were diluted 1:1,000 with 1% skim milk in PBS containing 1% Tween® 20 (Sigma-Aldrich) and incubated at 4°C overnight. Horseradish peroxidase-conjugated secondary antibodies in 2% skim milk were incubated with the blots for 1 h at room temperature. Immunoreactive proteins were detected using chemiluminescence reagent EzWestLumi Plus (ATTO) and visualized with a LAS-3000 instrument (Fujifilm, Tokyo, Japan).
Kidneys were fixed in 4% paraformaldehyde overnight and then embedded in paraffin. Kidney tissue sections were deparaffinized by three changes of Histoclear II (National Diagnostics, Atlanta, GA, USA) and then rehydrated in a graded series of ethanol. For antigen retrieval, the sections were heated in a TintoRetriever Pressure Cooker (Bio SB, Santa Barbara, CA, USA) with Borg Decloaker RTU (Biocare Medical, Pacheco, CA, USA) and were incubated with UV Hydrogen Peroxide Block (Thermo Fisher Scientific, Waltham, MA, USA). Sections were blocked with blocking solution for 1 h at room temperature, and then incubated overnight with the appropriate primary antibodies at 4°C. The primary antibodies used for immunostaining were anti-8-OHdG antibody (sc-66036, Santa Cruz) and anti-4 hydroxynonenal (ab48506, Abcam). Peroxidase activity was detected using the VECTASTAIN Elite ABC HRP Kit (PK-6200, Vector Labs, Burlingame, CA, USA) and VECTOR NovaRED Peroxidase (HRP) Substrate Kit (SK-4800, Vector Labs) according to the manufacturer’s instructions. Slides were mounted with VectaMount Permanent Mounting Medium (H-5000, Vector Labs) and analysed.
Mitochondrial ROS levels were assessed using MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen). MitoSOX™ Red is a nonfluorescent dihydroethidium-based dye that is oxidized by mitochondrial superoxide and emits a red fluorescence. IMCD cells were grown on glass coverslips in six-well plates. After 2 days of incubation, the cells were incubated with MitoSOX™ Red (5 μM) in HBSS/Ca/Mg (Gibco) for 10 min at 37°C and then washed three times with PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.2% Triton X-100 containing 1% bovine serum albumin for 10 min. Nuclei were stained with DAPI. The slides were mounted with fluorescence mounting medium (Dako, Santa Clara, CA, USA), and images were obtained under a confocal laser scanning microscope (LSM-700, Carl Zeiss, Oberkochen, Germany). The fluorescence intensity of MitoSOX™ Red was quantified using ImageJ software (NIH, Bethesda, MD, USA).
1mM stock solutions of MitoTracker™ Green (Thermo Fisher Scientific) were diluted with DMEM/F12 to a 200 nM working concentration and incubated with cells for 20 min at 37°C. After that, 1 ul of 5 mM MitoSOX™ Red were added and incubated for 10 min at 37°C.
IMCD cells (4×104 cells/well) were mixed with Matrigel (Corning) at a 1:1 ratio and plated in 8-well chamber slides. Culture medium was added after 1 h and replaced every day. Cells in Matrigel were grown in a humidified atmosphere containing 5% CO2 at 37°C for 4-6 days. Phase contrast images of individual cystic spheroids were obtained under a light microscope (IX70, Olympus, Tokyo, Japan) and ISP capture software (Olympus) after 4, 5, and 6 days in culture. The cyst area was measured using ImageJ software (NIH).
Mitochondria and the cytosol were separated by using the Mitochondria Isolation Kit (Pierce Biotechnology, Waltham, MA, USA) according to the manufacturer’s instructions. The mitochondrial fraction was confirmed using an anti-COX IV antibody (ab33985, Abcam). The cytosolic fraction was confirmed using an α-tubulin antibody (#3873, Cell Signaling).
Each experiment in this study was repeated at least three times. Data are means ± SD. Statistical analyses were performed with Student’s
All procedures conducted during this study were approved by the Sookmyung Women’s University School Institutional Animal Care and Use Committees.
Oxidative stress is known to contribute to ADPKD progression (Andries
The FOXO protein family is known to regulate cellular antioxidant defenses (Klotz
To identify the regulator of FOXO3a in ADPKD models, we performed an integrated analysis of miRNA sequencing data from
FOXO3 is involved in reactive oxygen metabolism by regulating mitochondrial gene expression (Ferber
We then investigated whether FOXO3 bound to specific DNA sequences of the
The effect of
It has been reported that oxidative stress appears in the early stages of ADPKD and worsens with disease progression (Andries
The respective roles of miR132 and Foxo3 in chronic kidney disease (CKD) following renal injury have been recently reported. In kidneys with ischemic injury, chronic hypoxia developed and it accumulated FOXO3 by inhibiting its degradation. In addition, hypoxia-activated HIF1α controlled the late phase of renal tubular repair by FOXO3 activation, which finally contributed to functional recovery (Li
FOXO3 is a negative regulator of mitochondrial ROS which mediates the defense against oxidative stress via regulation of ROS-scavenging enzymes (Liu
In our previous study, we reported the miRNAs that were differentially expressed in PKD model mice and human ADPKD patients by miRNA sequencing and microarray analysis, respectively, and reported that abnormal miRNA expression was closely associated with ADPKD (Li
To verify that miR-132-3p affected ROS accumulation, we assessed the expression of miR-132-3p in the ubiquinol-cytochrome c reductase binding protein (UQCRB) mutant cell line, which showed increased mitochondrial ROS (Kim
Based on the observed results of this study, we present a new mechanism of cystogenesis, in which increased miR-132-3p expression inhibits
This work was supported by grants from the Collaborative Genome Program for Fostering New Post-Genome Industry of the NRF [2014M3C9A2067613] and the Basic Science Program [2016R1A5A1011974].
The authors declare that they have no conflicts of interest.