Kidney specimens were collected from patients with DN or minimal change disease (Normal) who were diagnosed at The Second Xiangya Hospital of Central South University and used for subsequent RNA extraction and immunohistochemistry (IHC) staining. Patients aged ≥ 18 years with nephropathy due to diabetes type 1 or type 2 were included in this study and provided written informed consent. These patients did not receive any treatment previously. Patients with other diseases, such as other rental diseases, autoimmune diseases, cancers and cardiovascular diseases were excluded. Our study got approval from the Ethics Committee of The Second Xiangya Hospital of Central South University.
Cell culture and treatment
Human renal tubular epithelial HK-2 cells and HEK-293T cells were provided by the Cell Bank of Chinese Academy Sciences (Shanghai, China). HK-2 cells were treated with normal glucose (NG, 5.5 mM), high glucose (HG, 30.5 mM) or the osmotic control high mannitol (HM, 5.5 mM glucose and 25 mM mannitol) for 24, 48 or 72 h , which were used for subsequent analysis of HG-induced cell injury. Glucose (G7021) and mannitol (M4125) were obtained from Sigma (St. Louis, MO, USA). For 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or propranolol treatment, cells were treated with AICAR (S1802, Selleck, Houston, TX, USA) at 1 mM or propranolol (S4076, Selleck) at 100 µM.
MALAT1 and coding sequences of LIN28A and Nox4 were cloned into the pcDNA3.1 vector (V79020, ThermoFisher, Waltham, MA, USA) for overexpression of MALAT1 (oe-MALAT1), LIN28A (oe-LIN28A) and Nox4 (oe-Nox4). siRNA (10 nM) was selected for transient transfection thanks to its easy generation and introduction into cells with high efficiency. Specially, si-RNAs against MALAT1 (si-MALAT1), LIN28A (si-LIN28A) and Nox4 (si-Nox4) and scrambled negative control siRNAs (si-NC) were purchased from RiboBio (Guangzhou, China). HK-2 cells were transfected with vector, oe-MALAT1, si-MALAT1, oe-LIN28A, si-LIN28A, oe-Nox4, si-Nox4, si-LIN28A + vector, si-LIN28A + oe-Nox4, si-MALAT1 + vector, si-MALAT1 + oe-LIN28A or si-NC using the Lipo3000 reagent (L3000015, ThermoFisher) following the manual. Cells were harvested at 48 hours after transfection for subsequent assays. In some assays, cells were harvested and treated with NG or HG. For in vivo knockdown of MALAT1, viral vector-based shRNA with high transduction efficiency and long-term effect was used. Specially, the shRNA against MALAT1 (sh-MALAT1, Sigma) and scrambled shRNA (sh-NC, Sigma) were inserted into the pLKO.1 lentiviral vector (10878, Addgene, Watertown, MA, USA), sh-MALAT1 and sh-NC lentiviral particles were packaged in HEK-293T cells.
A rat model of DN
Sprague-Dawley male healthy rats (6-8-week-old) were provided by Hunan SJA laboratory animal Co., LTD (Changsha, Hunan, China) and blindly divided in to 4 groups: control, DN, DN + sh-NC and DN + sh-MALAT1. In the control group, rats were fed normally. In DN groups, rats were fed a high-glucose and fat diet for 8 weeks and intraperitoneally injected with streptozotocin (STZ, S0130, Sigma) at 55 mg/kg. Subsequently, the blood glucose was monitored every 3 days. DN was considered to be successfully established in rats which met following criteria: blood glucose > 16.7 mmol/L and 24 h urine volume and protein increased 150% after two weeks. DN rats were intravenously injected with 2×107 sh-NC or sh-MALAT1 lentiviral particles and fed a high-glucose and fat diet for 5 months. Finally, kidneys were harvested for subsequent assays. Animal procedures were approved by The Animal Care and Use Committee of The Second Xiangya Hospital of Central South University.
Real-time quantitative reverse-transcription PCR (qRT-PCR)
Tissues from patients and rats were homogenized. Total RNA was extracted from HK-2 cells and tissue homogenates using Trizol from Beyotime (R0016, Shanghai, China). Subsequently, RNA was quantified and reversely transcribed into cDNA. The expression of MALAT1, LIN28A and Nox4 was determined using quantitative PCR with SYBR Green (QPK-201, TOYOBO, Tokyo, Japan) and normalized to GAPDH. Gene expression was calculated using the 2−∆∆Ct method. Primers were listed in Table 1.
mRNA stability analysis in response to actinomycin D treatment
Actinomycin D, a widely used RNA transcription inhibitor for shutting off mRNA transcription, was used to investigate the decay rates of endogenous mRNAs. Briefly, HK-2 and HEK-293T cells were treated with actinomycin D (S8964, Selleck) at 5 µg/mL for indicated time. Subsequently, RNA was extracted and reversely transcribed into cDNA. The abundance of MALAT1 and Nox4 mRNA were analyzed by qRT-PCR.
MTT assay for cell viability
The culture medium was removed. 100 µL of fresh culture medium and 10 µL of MTT (M6494, ThermoFisher) were mixed well and added into each well. Cells were then incubated for 4 hours, and 50 µL of DMSO was added, and the absorbance (490 nm) was measured.
Hematoxylin and eosin (H&E), Masson and immunohistochemistry (IHC) staining
Kidney specimens from patients and rats were fixed, embedded and sliced into 5-µm sections. Slices were subsequently deparaffinized and rehydrated. H&E and Masson’s trichrome staining were applied to analyze renal histological change and collagen deposition. For IHC staining, antigen was retrieved in antigen retrieval solution, and slices were incubated with anti-LIN28A (1:50, ab175352, Abcam, Cambridge, UK) or anti-Nox4 (1:100, ab133303, Abcam). Subsequently, slices were incubated with an HRP-conjugated secondary antibody for 1 h. DAB (P0203, Beyotime) was used to visualize the signal. Slices were then stained with hematoxylin, mounted and imaged with a BX51 microscope from Olympus (Tokyo, Japan).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
The Click-iT TUNEL Assay kit (C10337) was obtained from ThermoFisher and used for analyzing cell apoptosis. HK-2 cells were fixed and permeabilized in 0.2% Triton X-100. Kidney tissues from rats were embedded in paraffin and sliced into 5-µm sections. Slices were subsequently deparaffinized, fixed and permeabilized in 0.2% Triton X-100. TdT rection was performed in cells and slices, and the Click-iT reaction was performed for detecting apoptotic cells. Cells and slices were stained with DAPI (C1005, Beyotime), mounted and imaged under a Leica confocal microscope (Wetzlar, Germany).
Enzyme-linked immunosorbent assay (ELISA)
The culture supernatants of HK-2 cells and rat serum were harvested and stored at -80˚C until use. The concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and albumin were determined by ELISA kits. Human and rat TNF-α (ab181421 and ab108913), IL-6 (ab178013 and ab100772) and albumin (ab108788 and ab108789) ELISA kits were obtained from Abcam.
Measurement of ROS
HK-2 cells were stained with using the fluorescent probes dihydroethidium (DHE; Invitrogen, Carlsbad, CA, MA) at 5 µM for 30 min at 37°C in the dark. DHE can be oxidized and then intercalates within DNA, staining the nucleus a bright fluorescent red. Subsequently, cells were washed with PBS for 10 min. Then, Intracellular ROS levels were measured using a fluorometer with excitation at 535 nm and emission at 610 nm (Varioskan™ LUX Multimode Microplate Reader; Thermo Fisher Scientific Inc.). Images were acquired at room temperature and DHE fluorescence intensity was measured using the ImageJ software (NIH, Bethesda, MD).
A drop of blood was collected from rat tails and the level of blood glucose was examined using the AlphaTRAK glucose meter (Zoetis, Parsippany, NJ, USA) following the manufacturer’s recommendation. Serum was prepared and urine was collected. The concentrations of blood urea nitrogen (BUN), serum and urinary creatinine were measured using the BUN colorimetric detection kit (EIABUN, ThermoFisher) and creatinine assay kit (ab65340, Abcam), respectively.
RNA immunoprecipitation (RIP)
HK-2 and HEK-293T cells were harvested and resuspend in nuclear isolation buffer (20 mM MgCl2, 40 mM Tris-HCl, 1.28 M sucrose and 4% Triton X-100). The nuclei were pelleted by centrifugation and resuspended in RIP buffer (5 mM EDTA, 0.5 mM DTT, 25 mM Tris, 150 mM KCl, 0.5% NP40, RNase and protease inhibitors). Chromatin was sheared, and the supernatants were collected by centrifugation. The anti-LIN28A (ab175352) and normal IgG control (ab37415, Abcam) were pre-coated on magnetic beads, mixed with the supernatants and incubated overnight. Subsequently, immunoprecipitated RNA was recovered using TRIzol, and the abundance of MALAT1 and Nox4 mRNA were analyzed by qRT-PCR.
RNA-Protein pull-down assay
HK-2 and HEK-293T cells were lysed, and cell lysates were harvested. Biotinylated MALAT1 or Nox4 RNAs (RiboBio) were added into cell lysates and incubated for 5 h at 4 ℃ for forming RNA-Protein complexes. Subsequently, streptavidin-conjugated magnetic beads (88816, ThermoFisher) were added, and samples were incubated for 2 h with gentle rotation at 4 ℃. The RNA-Protein complexes were pulled down by magnetic beads and eluted for western blotting analysis of LIN28A.
Rats were sacrificed, and kidneys were excised and weighed. Parts of kidneys were homogenized in lysis buffer. The supernatants of tissue homogenates and cell lysates were collected and quantified using the BCA kit (ab102536, Abcam). Protein (20 µg) was loaded each lane and electrophoresed prior to transfer to PVDF membranes (88518, ThermoFisher). Membranes were then blocked and incubated with anti-LIN28A (1:500, ab279647), anti-Nox4 (1:1000, ab133303), anti-AMPK (1:2000, ab80039), anti-p-AMPK (1:1000, 2531), anti-mTOR (1:1000, ab2732), anti-p-mTOR (1:500, 2971), anti-Bcl-2 (1:1000, ab196495), anti-Bax (1:500, ab32503) and anti-β-actin (1:6000, ab8227) overnight. Next day, membranes were incubated with an HRP-conjugated secondary antibody for 1 h. The bands were visualized using ECL substrate (1705061) from Bio-Rad (Hercules, CA, USA) and analyzed using the Image J software. Antibodies were from Abcam and Cell Signaling Technology (Danvers, MA, USA).
Data from at least three independent assays were expressed as mean ± standard deviation. The variance in two and multiple groups were analyzed by the Student's t test and one-way analysis of variance (ANOVA), respectively. P < 0.05 was statistically significant. *P < 0.05, **P < 0.01 and ***P < 0.001.