Human kidney tissue was obtained at the time of nephrectomy for conventional renal carcinoma and the clinical characteristics of the patients have been described before . The study was approved by the Nova Scotia Health Authority Research Ethics Board (Halifax, NS, Canada) and the Research Ethics Board of St Michael’s Hospital. A waiver of consent based on impracticability criteria was provided by the Nova Scotia Health Authority Research Ethics Board.
Podocin-cre+ (B6.Cg-Tg(NPHS2-cre)295Lbh/J) mice  were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and were bred with HotairLoxP/+ mice  obtained from H. Chang (Stanford University School of Medicine, Stanford, CA, USA) to generate Podocin-cre+Hotair+/+ (HotairCtrl) and Podocin-cre+Hotairfl/fl (HotairpodKO) mice. These mice were studied at 10–14 weeks of age under non-diabetic conditions. To measure glomerular Hotair expression in experimental DKD, C57BL/6 mice (The Jackson Laboratory) were rendered diabetic with i.p. injections of streptozotocin (STZ; 55 mg/kg in 0.1 mol/l citrate buffer, pH 4.5) after a 4 h fast for 5 consecutive days. Non-diabetic control mice received an equal volume of citrate buffer. Blood glucose was measured 12 days after the first i.p. injection and mice were followed for a further 12 weeks. Additionally, male db/m and db/db mice (The Jackson Laboratory) were studied at 20 weeks of age. To examine the effects of podocyte-specific Hotair knockout under diabetic conditions, male HotairCtrl and HotairpodKO mice were rendered diabetic with STZ as described above and were studied 12 weeks after the first i.p. injection of STZ (i.e. 10 weeks after confirmation of diabetes). Mouse phenotyping methods are described in electronic supplementary material (ESM) Methods. All experimental procedures adhered to the guidelines of the Canadian Council on Animal Care and were approved by St Michael’s Hospital Animal Care Committee.
Digoxigenin-labelled in situ hybridisation and RNAscope in situ hybridisation
Digoxigenin-labelled in situ hybridisation was performed on formalin-fixed, paraffin-embedded human or mouse kidney tissues as described in ESM Methods. RNAscope in situ hybridisation (Advanced Cell Diagnostics, Hayward, CA, USA) was performed according to the manufacturer’s instructions and using custom software as previously reported  and as described in ESM Methods.
Isolation of podocyte-enriched mouse glomeruli
Podocyte-enriched glomerular cell extracts were isolated from mouse kidneys using the CELLection Biotin Binder Kit (Thermo Fisher Scientific, Rockford, IL, USA) as described in ESM Methods.
For immunofluorescence microscopy, mouse slides were stained with antibodies in the following concentration: mouse nephrin 1:100 (AF3159, R & D Systems, Minneapolis, MN, USA), secondary antibody Alexa Fluor 488 donkey anti-goat 1:100 (Thermo Fisher Scientific). Co-staining was with the following antibodies: EZH2 1:100 (#5246, Cell Signaling Technology, Danvers, MA, USA), LSD1 1:100 (#2139, Cell Signaling Technology), H3K27me3 1:100 (#9733, Cell Signaling Technology), H3K4me1 (ab8895, Abcam, Cambridge, MA, USA), H3K4me2 1:100 (ab7766, Abcam), H3K4me3 1:100 (ab213224, Abcam), secondary antibody Alexa Fluor 647 donkey anti-rabbit 1:100 (Thermo Fisher Scientific). Slides were viewed using a Zeiss LSM 700 confocal microscope and analysis was conducted on approximately 30 podocytes from at least six glomeruli per slide using ImageJ 1.46r software (National Institutes of Health, Bethesda, MD, USA).
Transmission electron microscopy
Images of the entire glomerular profile were taken through three randomly selected glomeruli from 5–8 mice per group (Philips CM100, Electron Microscopy Research Services; Newcastle University, Newcastle, UK). Podocyte density was estimated by the method of Weibel and Gomez . Podocyte density was multiplied by mean glomerular volume (determined in periodic acid–Schiff [PAS] stained kidney sections ) to give an estimate of podocyte number per glomerulus. Glomerular basement membrane thickness and podocyte foot process width were measured in a masked manner in approximately ten micrographs (magnification ×5800) from each glomerular profile using ImageJ (National Institutes of Health).
Quantitative reverse transcription-PCR
Primers were obtained from Integrated DNA Technologies and had the following sequences: Hotair forward AGGGTCCCCAACATCGGTAGA, reverse TGCGGTGGAGATAGATGTGC or forward AGCTGAGAAGGCCTGAATGA, reverse AAGGGGTGAACAGTGATCTG; Rplp0 forward GCGTCCTGGCATTGTCTGT, reverse GAAGGCCTTGACCTTTTCAGTAAG; Rpl13a forward GCTCTCAAGGTTGTTCGGCTGA, reverse AGATCTGCTTCTTCTTCCGATA. Real-time quantitative reverse transcription- PCR (qRT-PCR) was performed on a Viia 7 real-time PCR system (Thermo Fisher Scientific) using SYBR Green and data analysis was performed using the comparative Ct method.
Conditionally immortalised mouse podocytes
Differentiated, conditionally immortalised mouse podocytes were cultured as previously described . Cells were serum starved for 4 h and incubated in normal media (5.6 mmol/l glucose) or in 25 mmol/l glucose (high glucose) for 48 h. Mannitol (19.4 mmol/l) added to normal media served as the osmotic control. For p65 knockdown, cells were transfected with 50 nmol/l mouse sequence-specific short interference RNA (siRNA) for p65 (sc-29411, Santa Cruz Biotechnology, Dallas, TX, USA) or scrambled siRNA (AM4611, Thermo Fisher Scientific) for 4 h prior to exposure to high glucose for 48 h. Chromatin immunoprecipitation by RNA purification (ChIRP) was performed using an EZ-Magna ChIRP Interactome Kit (EMD Millipore, Etobicoke, ON, Canada). Biotinylated Hotair probes were designed and synthesised by Biosearch Technologies (Petaluma, CA, USA) to cover the entire length of Hotair (ESM Table 1). The LacZ probeset was from Millipore. Primer sequences for Hotair targets (Hoxd1, Hoxd3, Hoxd11, Dlk1, Plag1, Dcn and H19) and control genes (Vamp5, Emp2) were as reported in  and were from Integrated DNA Technologies (Coralville, IA, USA). Values were normalised relative to LacZ binding compared with input DNA . Chromatin immunoprecipitation (ChIP) was performed using a Magna ChIP kit (EMD Millipore) with an antibody against p65 (1:100 dilution; #8242, Cell Signaling Technology) or an equal concentration of normal rabbit IgG control (sc-2027, Santa Cruz Biotechnology). qRT-PCR was performed using primers specific for the mouse Hotair promoter (forward CCCAGCCAGGTAGGTAGAGT, reverse GAAGGGGCTGATGGATGCTT).
was performed with antibodies in the following concentrations: nephrin 1:1000 (AF3159, R & D Systems), p65 1:100 (#8242, Cell Signaling Technology), β-actin 1:10,000 (#A1978, Sigma-Aldrich, Oakville, ON, Canada). Densitometry was performed using ImageJ.
Mesangial matrix index
Mesangial matrix accumulation was calculated in approximately 30 glomeruli from PAS-stained kidney sections by investigators masked to the study groups, as previously described .
Immunostaining of mouse kidney tissue was performed as previously described  with the following antibodies: collagen IV 1:500 (AB756P, EMD Millipore), α-smooth muscle actin (α-SMA) 1:400 (ab5694, Abcam), nephrin 1:200 (AF3159, R&D Systems). The proportion of glomerular area positively immunostaining was determined in 30 random glomerular profiles in each kidney section using ImageScope for collagen IV and α-SMA and HALO version 2.3.2089.23 (Indica Labs, Corrales, NM, USA) for nephrin immunostaining.
Transcriptomic data were extracted from the NCBI Gene Expression Omnibus (GEO) database, accessible under the series number GSE66494. The details of the dataset are described in . In brief, we studied the ‘discovery cohort’ consisting of transcriptomic data derived using the Agilent Whole Human Genome Microarray 4x44K from biopsy tissue from 48 patients with chronic kidney disease (CKD) compared with tissue from five control participants. Differential expression of HOTAIR, HOXC11, TGF-β1 (also known as TGFB1), CTGF (also known as CCN2) and CCL2 was assessed using limma, an R/Bioconductor software package. The false discovery rate was controlled using the Benjamini–Hochberg method. A p value of ≤0.05 was used to identify differentially expressed genes. Pearson correlation coefficient was calculated using Microsoft Excel. Boxplots and heatmaps were generated using limma.
Data are expressed as means ± SD. Statistical significance was determined by one-way ANOVA followed by Fisher least significant difference post hoc test for multiple groups comparison or by two-tailed Student’s t test for comparison between two groups, unless otherwise stated. All statistical analyses were performed using GraphPad Prism 6 for Mac OS X (GraphPad Software, San Diego, CA, USA) unless otherwise stated.