RNA-Seq Reveals Altered Gene Expression Levels in Proximal
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www.nature.com/scientificreports OPEN RNA‑seq reveals altered gene expression levels in proximal tubular cell cultures compared to renal cortex but not during early glucotoxicity Linnéa M. Nilsson1, Miguel Castresana‑Aguirre 2, Lena Scott3 & Hjalmar Brismar 1,3* Cell cultures are often used to study physiological processes in health and disease. It is well‑known that cells change their gene expression in vitro compared to in vivo, but it is rarely experimentally addressed. High glucose is a known trigger of apoptosis in proximal tubular cells (PTC). Here we used RNA-seq to detect diferentially expressed genes in cultures of primary rat PTC, 3 days old, compared to cells retrieved directly from rat outer renal cortex and between PTC exposed to 15 mM glucose and control for 8 h. The expression of 6,174 genes was signifcantly up- or downregulated in the cultures of PTC compared to the cells in the outer renal cortex. Most altered were mitochondrial and metabolism related genes. Gene expression of proapoptotic proteins were upregulated and gene expression of antiapoptotic proteins were downregulated in PTC. Expression of transporter related genes were generally downregulated. After 8 h, high glucose had not altered the gene expression in PTC. The current study provides evidence that cells alter their gene expression in vitro compared to in vivo and suggests that short‑term high glucose exposure can trigger apoptosis in PTC without changing the gene expression levels of apoptotic proteins. Cell cultures, both primary and immortalized, are ofen used as models in biological research to investigate physi- ological processes in health and disease. It is commonly known that cells change their gene expression in vitro compared to in vivo. Yet the extent of the diferentiation is not fully understood. We therefore wanted to compare gene expression in primary cell cultures, 3 days old, with cells retrieved directly from the kidney. Our group has previously used primary rat proximal tubule cells (PTC) to investigate the intrinsic apoptotic pathway in diseases such as proteinuric kidney disease 1 and hemolytic uremic syndrome 2. In this study, we chose to examine changes in gene expression between primary PTC and tissue from outer renal cortex, which volume mostly consists of proximal tubule3. Te main aim of this study was to discern in which groups of genes that the gene expression level is most altered in primary PTC cultures compared to cells in the outer renal cortex. In addition, we wanted to investigate if gene expressions related to our previous studies had signifcantly changed within 3 days. Hyperglycemia is one of the most common symptoms in diabetic kidney disease, where PTC have been identi- fed as one of the targets of glucotoxicity 4. Our group have previously used PTC to study the apoptotic response to short-term high glucose exposure 5. In addition to study changes in gene expression in PTC compared to outer renal cortex, we therefore decided to study whether gene expression levels would change in PTC exposed to 15 mM glucose for 8 h compared to control. If that was the case, we wanted to identify which groups of genes had changed their expression the most. 1Science for Life Laboratory, Department of Applied Physics, Royal Institute of Technology, Box 1031, 171 21 Solna, Sweden. 2Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden. 3Science for Life Laboratory, Department of Women’s and Children’s Health, Karolinska Institutet, Solna, Sweden. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:10390 | https://doi.org/10.1038/s41598-020-67361-3 1 Vol.:(0123456789) www.nature.com/scientificreports/ Methods Cell culture and tissue preparation. Twenty-day-old male Sprague Dawley rats were used for prepa- ration of proximal tubule slices and PTC cultures. All animals were housed under controlled conditions of light and dark (12:12 h) and given a standard diet containing 20% protein by weight and tap water were avail- able ad libitum. All experiments were performed according to Karolinska Institutet regulations concerning care and use of laboratory animals and were approved by the Stockholm North ethical evaluation board for animal research. Proximal tubule slices were collected from the outer 150 µm of the renal cortex, where 90% of the tubular volume is proximal tubules3. Primary cultures of rat PTC were prepared as previously described1 using the outer 150 µm renal cortex as starting material. Cells were seeded in 60-mm wells and cultured in 37 °C at an approxi- mate humidity of 95–98% with 5% CO 2 for 3 days before experiments. Culture medium was changed every 24 h. Cells were exposed to 15 mM glucose (HG) or 5.6 mM glucose (control) for 8 h. Kidney cortex samples were prepared in replicates from three animals. PTC samples were prepared from three separate cultures and pairwise exposed to HG or control. RNA‑seq. Cells and tissue samples were collected and mRNA extracted and purifed with RNeasy mini kit (cat. no. 74134, Qiagen AB, Sollentuna, Sweden) following manufacturer’s instructions. Te quality of the start- ing RNA was validated with an Agilent Bioanalyzer before cDNA libraries were created. Te cDNA libraries were created by National Genomics Infrastructure at Science for Life Laboratory (Solna, Sweden) using Illumina TruSeq Stranded mRNA with poly-A selection. Each sample was used to generate two separate cDNA libraries. Quality controls of the libraries were performed by National Genomics Infrastructure at Science for Life Labora- tory (Solna, Sweden) using MultiQC. Bioinformatics analyses. Diferential expression analysis was performed with the R package edge R6 and heatmaps were created using the R package pheatmap. Te cDNA libraries were aligned to a reference genome, which was created using the Rattus norvegicus genome from National Center for Biotechnology Information webpage7. Te annotations for each gene was retrieved from National Center for Biotechnology Information webpage7 and matched to each gene start and stop codon position. Te gene symbols were added from the R package org.Rn.eg.db8. Gene symbols occasionally appeared in the list of genes more than once. Only the gene transcript with the highest number of counts for each gene was saved. Te list of genes was fltered with the edgeR function flterBy- Expr. We required the genes to have at least 10 counts in one sample and at least a total of 20 counts across all samples to be included in the analysis. Tese requirements were fulflled by 7,615 genes. We performed trimmed mean of M-value normalization to remove possible composition bias between samples. Diferences between the expression profles of the samples were visualized with a multi-dimensional scaling plot (Fig. 1a). Te plot shows a large diference in gene expression profle between renal cortex and PTC and a small diference between PTC incubated in control and HG medium for 8 h. Te fold-change (FC) between renal cortex and PTC was ~ 26 = 64 and the FC between control and HG exposed PTC was within 20.5 ≈ 1.4, except for one HG sample where the FC was ~ 21.5 ≈ 2.8 from control samples. Te common negative binomial dispersion among the samples was estimated to approximately 0.023 and the biological coefcient of variation is shown in Fig. 1b. Bax and Bcl‑xl abundance assessment. Abundance of Bax and Bcl-xL was assessed as previously described5. Briefy, 3 days old PTC were incubated with control or HG for 8 h. Cells were fxed with 4% para- formaldehyde (pH 7.4) for 10 min, permeabilized with 0.3% Triton X-100 for 10 min and blocked with 5% BSA in 0.1% Triton X-100 for 1 h. Primary antibodies mouse monoclonal anti-Bax (6A7) (5 μg/ml) (Abcam, Cam- bridge, UK) and rabbit monoclonal anti-Bcl-xL (54H6) (1:200) (Cell Signaling Technology, Danvers, MA, USA) were applied over night at 4 °C. Cells were washed and secondary antibodies Alexa Fluor 546 goat anti-mouse IgG (Life Technologies, Carlsbad, CA, USA) and Alexa Fluor 546 goat anti-rabbit IgG (Life Technologies, Carls- bad, CA, USA) were applied for 1 h at room temperature. Secondary antibody controls were subjected to the same treatment, but primary antibodies were omitted. Cells were imaged with a Zeiss LSM 510 confocal micro- scope equipped with × 63/1.4 NA oil objective. Te microscope setting was kept fxed for all measurements. Te Bax and Bcl-xL abundances were analyzed in Matlab (Te MathWorks, Natick, MA, USA). Te total abundance of Bax and Bcl-xL was calculated as the percentage of Bax or Bcl-xL (pixels) normalized to cell size (pixels). On each coverslip, at least three cells were analyzed. Te control group was set to 100%. Statistics. Statistical signifcance of the diferential expression analysis was determined with a one-way ANOVA for each gene using the glmQLFTest function in edge R. Te signifcance of diferentially expressed genes was determined by false discovery rate (FDR). A FDR < 0.05 was considered signifcant. Results Mitochondrial and metabolism GO terms were most altered in PTC compared to renal cor‑ tex. We frst screened for diferentially expressed genes in PTC cultures compared to outer renal cortex slices. Te expression of 3,042 genes was signifcantly downregulated in PTC compared to renal cortex and it was signifcantly upregulated for 3,132 genes. To identify the groups of genes that were overrepresented in PTC com- pared to renal cortex we performed a gene ontology (GO) enrichment analysis. Mitochondrial and metabolism related GO terms, including mitochondrion (GO-CC:0005739), oxidation–reduction process (GO-BP:0055114) SCIENTIFIC REPORTS | (2020) 10:10390 | https://doi.org/10.1038/s41598-020-67361-3 2 Vol:.(1234567890) www.nature.com/scientificreports/ Figure 1.