DOI: 10.5061/dryad.v9s4mw6rb
title: RNA-seq reveals altered gene expression levels in proximal tubular cell cultures compared to renal cortex but not during early glucotoxicity

description: AbstractCell 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 differentially
 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 significantly 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.
description: <strong>Cell culture and tissue preparation</strong> Twenty-day-old male Sprague Dawley rats were used for preparation of proximal tubule slices and PTC cultures. All animals were housed under controlled conditions of light and dark (12:12 hours) and given a standard diet containing 20 % protein by weight and tap water were available <i>ad libitum</i>. 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 tubules (3). Primary cultures of rat PTC were prepared as previously described (1) using the outer 150 µm renal cortex as starting material. Cells were seeded in 60-mm wells and cultured in 37°C at an approximate humidity of 95-98 % with 5 % CO<sub>2</sub> for three days before experiments. Culture medium was changed every 24 hours. Cells were exposed to 15 mM glucose (HG) or 5.6 mM glucose (control) for 8 hours. 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.  <strong>RNA-seq </strong> Cells and tissue samples were collected and mRNA extracted and purified with RNeasy mini kit (cat. no 74134, Qiagen AB, Sollentuna, Sweden) following manufacturer’s instructions. The quality of the starting RNA was validated with an Agilent Bioanalyzer before cDNA libraries were created. The 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 Laboratory (Solna, Sweden) using MultiQC.
description: The data is stored as tar archives. Each tar file contains FASTQ
 RNAseq data organised according to treatment P10265_101
 renal cortex P10265_102 renal cortex
 P10265_103 renal cortex P10265_105 PTC
 cultured 3 days P10265_106 PTC cultured 3
 days P10265_202 PTC cultured 3 days
 P10265_108 PTC cultured 3 days High Glucose
 P10265_109 PTC cultured 3 days High Glucose
 P10265_203 PTC cultured 3 days High Glucose