RNA-seq reveals altered gene expression levels in proximal tubular cell cultures compared to renal cortex but not during early glucotoxicity

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.
Methods
Cell culture and tissue preparation

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 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 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 % CO2 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. 

RNA-seq 

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.