Diabetes

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

Cell models are often used to study signaling pathways in health and disease in a controlled and isolated environment. It is commonly known, but rarely discussed, that cells may change their gene expression in vitro compared to in vivo. Here we addressed this question using RNA-seq to identify differentially expressed genes between renal outer cortex and 3 days old PTC cultures.

The gene expression level of most genes, 6,174 of 7,615, was significantly altered in the PTC cultures compared to the renal outer cortex slices. We identified the largest changes in gene expression levels in mitochondrial and metabolism related GO terms, which could be indicating that cells in culture have a changed energy expenditure and metabolism compared to cells in the renal cortex. It is known that cells gradually lose their phenotype in vitro. In the current study, the primary cells still expressed the same genes as the renal cortex slices even though the expression level for a large number of genes was significantly up- or downregulated. These data indicate that the differentiation process of cells starts directly after cells are isolated. Primary cells are generally believed to keep their gene expression compared to immortalized cells. The results from the current study therefore opens up questions about to which extent the gene expression of immortalized cells differ from primary cells and from their original tissue. These questions still remain to be determined.

It has previously been shown that the expression of many proteins change when podocytes are cultured in vitro9. In particular, it has been demonstrated that the culture conditions, such as the elastic modulus of the substrate, have a strong influence on the expression of proteins related to the actin cytoskeleton, including stress fibers and focal adhesion proteins10. In the current study we found that the expressions of cytoskeleton genes were significantly altered in cultures of PTC. This may be an indication that an altered environment initiates a change in PTC morphology, which could be a consequence of PTC losing their polarization in vitro compared to in vivo.

Apoptosis associated GO terms were overrepresented in PTC compared to renal cortex. Differentially expressed genes in the overrepresented apoptosis related GO terms included genes that codes for proteins involved in fibrosis, apoptosis and inflammation. The gene expression of the antiapoptotic proteins Bcl2 (Bcl2) and Bax inhibitor 1 (Tmbim6) were significantly downregulated in PTC and the gene expression of the executor protein caspase 2 (Casp2) was significantly upregulated, suggesting that PTC may have an increased susceptibility to apoptosis in vitro compared to proximal tubules in vivo. The gene expression for the TGFβ superfamily members bone morphogenetic proteins 1, 3 and 5 (Bmp1/3/5) were both up- and downregulated in PTC compared to tubule and the gene expression of caspase 12 (Casp12) was significantly upregulated. These data suggest a change in fibrosis and inflammation processes, which could be an indication that a healing process has started due to the dissociation of cells in culture compared to in vivo, where cells are less spread out.

The gene expression of many transporters was significantly up- or downregulated in PTC compared to renal cortex, including the gene expressions for the sodium-dependent glucose transporters NaGLT (Naglt), SGLT1 (Slc5a1) and SGLT2 (Slc5a2), which were downregulated, and the gene expression for the glucose transporter GLUT1 (Slc2a1), which was upregulated. The gene expression for GLUT2 (Slc2a2) was downregulated in PTC compared to renal cortex. We also found that several genes belonging to the solute carrier membrane group (SLC) of transport proteins relevant for transport of other substrates, including amino acids, fatty acids and ions, were also significantly changed (Table 8). Altogether these results indicate that cells in culture change their transport of solutes. In particular is the glucose uptake affected in PTC with a reduced sodium-dependent glucose uptake compared to the cells that express sodium-glucose cotransporters in vivo, in order to accommodate for a lower rate of glucose metabolism.

Table 8 The top 15 differentially expressed genes in the Slc family in PTC compared to renal cortex.

In addition to the downregulation of sodium-dependent glucose transporters, the gene expression of aquaporins (Aqp1/2/3/6), Na+/K+-ATPase subunit α1 (Atp1a1) and β1 (Atp1b1) were also significantly downregulated. SGLT 1 and 2, aquaporins and Na+/K+-ATPase are transporters that in vivo facilitates vectorial transport across PTC. In vitro, PTC are no longer polarized, which may contribute to decreased gene expression of transporters that facilitates vectorial transport.

RNA-seq has previously been used to study gene expressions of the whole kidney10,11. To focus the current study on the proximal tubule we instead used thin slices of outer renal cortex. A large number of cell-type specific genes11 that are expressed in other kidney cells than PTC were therefore not expressed in our samples. A drawback of the current study is that only 90% of the renal outer cortex slices consist of proximal tubular volume3, whereas 99% of PTC are SGLT2-positive when stained with antibodies1. The fraction of PTC is thereby higher in PTC samples compared to the renal tissue samples and may have affected the readout of the GO enrichment analysis and differential expression analysis between cells and tissue, especially for PTC specific genes. Differences in kidney cell composition, PTC-specific genes and PTC segment specific genes among the samples is shown in Fig. 3, SI Figs. S1, S2, S3 and S4, respectively, using cell-type and segment specific genes (SI Tables S4S4)11,12. To fully conclude differentially expressed genes in PTC cultures a tissue sample containing only proximal tubule would be required.

Figure 3
figure3

Heatmap for cell-type specific markers10. We compared 3 days old cell cultures of primary PTC with cells retrieved directly from the outer renal cortex, and between PTC exposed to 15 mM of glucose and control for 8 h. Genes were clustered by cell-type. Gene expression goes from blue (low expression) to red (high expression). Samples are ordered by days in culture (DaysInCulture), and by control and high glucose (HG).

Other studies have shown that hyperglycemia exerts a change in gene expressions of PTC after 24–48 h of exposure to 25–30 mM glucose4,13. Apoptosis related genes were significantly altered after exposure to 25 mM glucose for 48 h. The current study did not find a significant change in gene expression. This is likely due to a shorter exposure to HG. However, the present study only shows a snapshot of what happens after 8 h of glucose exposure. It is therefore not possible to conclude what happens before or after 8 h. To determine how HG effects the gene expression levels in PTC, a time or does response curve might be necessary. The protein expression of the antiapoptotic protein Bcl-xl was however significantly downregulated after 8 h of exposure to HG, while the protein expression of the proapoptotic protein Bax was significantly upregulated (Fig. 4). These data suggest that HG triggers an acute regulation of apoptotic protein levels within 8 h, without regulation of gene expression, which has been reported to be altered after 48 h4.

Figure 4
figure4

Data revised from our publication Nilsson et al.5. Left: representative images of immunostaining for Bax and Bcl-xl in PTC incubated with control or HG containing medium for 8 h. Scale bars are 10 µm. Each image visualizes one cell. Right: quantification of Bax and Bcl-xl protein expression in PTC incubated with control or HG containing medium for 8 h. Data is expressed as mean ± SEM. n = 15 coverslips from 5 individual cell preparations. **p < 0.01, ***p < 0.001.

One potential drawback of the current study is that one of the HG samples differed somewhat in FC. The reason for this variation is not known. It may be within the expected biological variation, since primary cells could respond differently to HG exposure. To full conclude how short-term exposure to high glucose affects the gene expression levels it might be necessary to include more than three replicates to better estimate the effects of the biological variance.

In the current study we conclude that genes are differentially expressed in cultured cells compared tissue, which highlights the importance to verify that cells still express the genes of interest when setting up experiments. The results from this study show that PTC still express the same genes as in tubules, but that the gene expression level is altered. Short-term exposure of HG did not significantly alter gene expression levels, which may be a later response to glucotoxicity.

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