Hypoxia-induced long non-coding RNA Malat1 is dispensable for renal ischemia/reperfusion-injury

Renal ischemia-reperfusion (I/R) injury is a major cause of acute kidney injury (AKI). Non-coding RNAs are crucially involved in its pathophysiology. We identified hypoxia-induced long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) to be upregulated in renal I/R injury. We here elucidated the functional role of Malat1 in vitro and its potential contribution to kidney injury in vivo. Malat1 was upregulated in kidney biopsies and plasma of patients with AKI, in murine hypoxic kidney tissue as well as in cultured and ex vivo sorted hypoxic endothelial cells and tubular epithelial cells. Malat1 was transcriptionally activated by hypoxia-inducible factor 1-α. In vitro, Malat1 inhibition reduced proliferation and the number of endothelial cells in the S-phase of the cell cycle. In vivo, Malat1 knockout and wildtype mice showed similar degrees of outer medullary tubular epithelial injury, proliferation, capillary rarefaction, inflammation and fibrosis, survival and kidney function. Small-RNA sequencing and whole genome expression analysis revealed only minor changes between ischemic Malat1 knockout and wildtype mice. Contrary to previous studies, which suggested a prominent role of Malat1 in the induction of disease, we did not confirm an in vivo role of Malat1 concerning renal I/R-injury.


Ischemic/Reperfusion injury Protocol
Clamping of renal pedicles was applied to induce significant renal I/R injury as described previously 1 . Following isoflurane anaesthesia male C57BL/6 mice were subjected to median laparotomy, thereafter renal pedicles were dissected and a vascular clamp was applied for 30 minutes.
For the survival analysis sham operated animals (n = 4) were also included. Blood samples for analysis of renal function parameters (serum-urea and -creatinine) were drawn on days 0, 1, 3 and 7 (analysed on a Beckman Analyzer, Beckman Instruments GmbH, Munich, Germany). In a second group of mice the renal pedicle was only clamped on the left side (unilateral I/R-injury). In this setting the contralateral kidney serves as an internal control to the injured kidney (I/R-kidney). These animals were sacrificed on day 1 and day 7 after renal IR injury and kidneys were harvested for further examination. At each time point 6 mice were used for unilateral I/R-injury. In bilateral I/R-injury studies 10 mice were used per group. In vivo studies conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).

Histology, Immunostaining
After kidney extraction, a representative part of each kidney was fixed immediately in PBS-buffered 4% paraformaldehyde and embedded in paraffin. Certain immunostainings were also performed in cryosections. Four-micrometer sections were used for immunostaining and for hematoxylin/eosin staining to evaluate histologic damage.
The severity of morphologic renal damage was assessed in a blinded manner using an arbitrary score based on PAS-stained kidney sections following a modification of a protocol developed by Broekema et al 2 . Briefly, the extent of four typical I/R injuryassociated damage markers (i.e., dilatation, denudation, intraluminal casts, loss of brush border membrane and cell flattening) was expressed in arbitrary units (AU) in a range of 0 to 4 according to the percentage of damaged tubules: 0, no damage; 1, less than 25% damage; 2, 25%-50% damage; 3, 50%-75% damage; and 4, more than 75% damage. Immunostainings for inflammatory cell influx was performed using the following primary antibodies: monoclonal rat anti-mouse F4/80 (Serotec, Oxford, United Kingdom), monoclonal rabbit anti-Mouse CD3 (Abcam, Cambridge, UK).
Analyses of capillary rarefaction in outer medulla were evaluated after fluorescent immunohistochemical staining for polyclonal rabbit anti-mouse CD31 (Abcam, Cambridge, UK). Immunostainings for detection of proliferating cells was performed using the following primary antibody: polyclonal rabbit anti-mouse Ki67 (Abcam, Cambridge, UK). Deparaffinized kidney sections were boiled in citrate buffer for antigen retrieval, blocked with 5% milk, and incubated overnight at 4°C with primary antibodies. This was followed by antibody visualization using Alexa 594 secondary antibodies (Molecular Probes/Invitrogen, Carlsbad, CA). Quantification of F4/80-, CD3-, CD31-and Ki67 expressing cells was done by counting of positive cells in five randomly chosen, non-overlapping fields in outer medulla.

HK-2 cells were cultured in DMEM medium supplemented with 10% FCS and 1%
Penicillin/Streptomycin. Cells were grown to 60% to 70% confluence and used for further downstream analyses. Cells were subjected to hypoxia (0.1% O2) for 24 hours and reoxygenation for 2 hours. Apoptosis was determined by Caspase 3/7 Glo assay according to the manufactures instruction. Briefly, this assay is luminescent based measuring the caspase-3 and -7 activities in cultured adherent cells. The proluminescent caspase-3/7 substrate consists of the tetrapeptide sequence DEVD. If this substrate is cleaved, aminoluciferin is released to produce light. In addition, Annexin Apoptosis Assay was performed according to the manufactures instruction (FlowCellectTM Annexin Red Kit, Millipore). Briefly, cells undergoing early apoptosis were detected with Annexin V+ and 7-amino actinomycin D (7-AAD)-stainings using fluorescence-activated cell sorting (FACS) on a Guava easyCyteTM sorter (Millipore) using Cytosoft software (Guava Technologies) according to the manufacturer's instructions.

Protein Analysis
Western blot analysis was performed using 10 to 40 μg of total protein. Cell lysis was performed (cell lysis buffer, Cell Signaling Technology) and protein electrophoresis was initiated. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% milk in TBS-Tween, and probed overnight at 4°C with the following primary antibodies: Anti-HIF-1α antibody (Abcam) and Anti-β-Actin (Sigma-Aldrich) was used as an internal loading control and for normalization of protein quantification. Immunoblots were scanned and quantified using ImageJ densitometry software.

Subcellular fractionation of cells
To determine subcellular localization of Malat1 we employed a protocol described previously by Cabianca and co-workers 3 . Here, the cytoplasmic fraction was separated from the nuclear-soluble and nuclear chromatin associated fraction. Briefly, cells trypsinized and centrifuged. The pellet was lysed with 175 μl of cold RLN1 solution (50 mM Tris HCl pH 8.0; 140 mM NaCl; 1.5 mM MgCl2; 0,5% NP-40; 2mM Vanadyl Ribonucleoside Complex; Sigma) and incubated for 5 min on ice. Next, the suspension was centrifuged at 4°C and 300 g for 2 min and the supernatant, corresponding to the cytoplasmic fraction, was transferred into a new tube and stored on ice. The pellet containing nuclei was extracted with 175 μl of cold RLN2 solution (50 mM Tris HCl pH 8.0; 500 mM NaCl; 1.5 mM MgCl2; 0,5% NP-40; 2mM Vanadyl Ribonucleoside Complex) and incubated for 5 min on ice. The suspension was centrifuged at 4°C and 16360 g for 2 min and the supernatant, corresponding to the nuclear-soluble fraction, was transferred into a new tube and stored on ice. The remaining pellet corresponds to the chromatin-associated fraction. Cytosolic expression might suggest a function as competing endogenous RNA regarding sponging of microRNAs, while nuclear expression suggests interaction with chromatin modifying factors or transcription factors.

Chromatin immunoprecipitation (ChIP)
ChIP was performed using the MAGnify Chromatin Immunoprecipitation System (Thermo Fisher Scientific) according to the manufacturer' instructions. Cells (1 × 10 7 ) were cross-linked in 1% formaldehyde for 10 min. After cell lysis, the chromatin was sheared using a sonicator to an average size of 500 bp and enriched with an antibody against HIF-1α or with isotype IgG coupled to magnetic Dynabeads (Invitrogen) at 4°C overnight. Subsequently, crosslinks were reversed, the DNA cleaned by RNase A (0.2 mg/mL) and proteinase K (2 mg/mL) and purified by phenol/chloroform. The specific sequence from immunoprecipitated and input DNA were determined by PCR primers for Malat1 promoter upstream region (see primer list in Supplemental Table 3).

Cell cycle analysis
To detect cells in the different phases of the cell cycle, cellular DNA was labeled with propidium iodide using Guava Cell Cycle Reagent (Guava Technologies). Labeled cells were analysed using fluorescence-activated cell sorting (FACS) on a Guava easyCyte sorter (Millipore) using the Cytosoft software (Guava Technologies) according to the manufacturer's instructions.

Proliferation Assay
To measure the proliferation rate in vitro, Bromodeoxyuridine (BrdU) Cell Proliferation ELISA Kit (Abcam) was used according to the manufacturer's instructions.

Ex vivo cell purification
The cellular origin of Malat1 following induction of I/R-injury was investigated by fluorescence-associated cell sorting (FACS) analysis using specific antibodies following a protocol by Chau et al. with modifications 1,4 . Following clamping of the right renal pedicle and a reperfusion period of 24 hours both kidneys were extracted, decapsulated, homogenized, then incubated at 37ºC for 30 min with Collagenase II (81 U/ml) and DNase (100U/ml, Roche) in serum free DMEM. After centrifugation, cells were re-suspended in 5ml of PBS/1% BSA, and filtered (40μm). Cells were separated using the following specific antibodies or lectins: rat anti-mouse-CD31-PE (1: Subsequently, RNA was isolated by Trizol method.

Scratch wound healing assay
Transfected HUVECs were cultivated in Endothelial Basal Medium with supplements at 37°C, 5% CO2. The scratches in the cell monolayer were generated with a 100-μl tip, and the cells were photographed at 0, 8, and 24 hours with a Nikon Ti 90 microscope (Germany). Subsequently, the cell free area was calculated.

Reverse Transcription-Polymerase Chain Reaction, and Global Transcriptome
Analysis RNA isolation was performed with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction analysis was performed in an ICycler (Bio-Rad). Gene array analysis was performed with the Affymetrix GeneChip system according to the manufacturer's instructions (Affymetrix Systems). Next Generation Small RNA Sequencing on an Ilumina platform was performed at Helmholtz Zentrum for Infection Biology, Braunschweig, Germany. A total of 24,410 protein-coding genes and 564 small RNAs were detected in our samples.
Reverse transcription was performed with total RNA using oligoDT primers (Bio-Rad).
Amplified cDNA was used as a template for quantitative PCR. Reverse transcription polymerase chain reaction analysis was performed in an ICycler (Bio-Rad) with SYBRgreen mastermix. The specific cel-miR-39 primer was purchased from Thermo Fisher Scientific. A list of primers used can be found in Supplemental Table 3.

Transfection Assays
Transient liposomal transfection of antisense oligonucleotides targeting Malat1 Cells were incubated for 4 hours before the media were changed to fresh media.