Acidosis Activates the Nrf2 Pathway in Renal Proximal Tubule-Derived Cells through a Crosstalk with Renal Fibroblasts

Abstract

Crosstalk of renal epithelial cells with interstitial fibroblasts plays an important role in kidney pathophysiology. A previous study showed that crosstalk between renal epithelial cells and renal fibroblasts protects against acidosis-induced damage. In order to gain further mechanistic insight into this crosstalk, we investigated the effect of acidosis on the transcriptome of renal epithelial cells (NRK-52E) and renal fibroblasts (NRK-49F) in co-culture by RNASeq, bioinformatics analysis and experimental validation. Cells were exposed to acidic media or control media for 48 h. RNA and protein from whole cell lysate were isolated. In addition, cells were fractionated into cytosol, nucleus and chromatin. RNASeq data were analyzed for differential expression and pathway enrichment (ingenuity pathway analysis, IPA, QIAGEN). Total and phosphorylated protein expression was assessed by Western blot (WB). Transcription factor activity was assessed by luciferase reporter assay. Bioinformatic analysis using differentially expressed genes according to RNASeq (7834 for NRK-52E and 3197 for NRK-49F) predicted the antioxidant and cell-protective Nrf2 pathway as acidosis-induced in NRK-52E and NRK-49F cells. Activation of Nrf2 comprises enhanced Nrf2 phosphorylation, nuclear translocation, DNA binding and initiation of a cell protective transcriptional program. Our data show that acidosis enhances chromatin-associated Nrf2 expression and the abundance of phosphorylated Nrf2 in the chromatin fraction of NRK-52E cells in co-culture but not in monoculture. Furthermore, acidosis enhances the activity of a reporter for Nrf2 (ARE-luciferase). Despite the bioinformatics prediction, NRK-49F cells did not respond with Nrf2 activation. Transketolase (TKT) is an important regulator of antioxidant and homeostatic responses in the kidney and a canonical Nrf2 target gene. We show that protein and mRNA expression of TKT is increased in NRK-52E cells under co-culture but not under monoculture conditions. In conclusion, our data show that extracellular acidosis activates the cytoprotective transcription factor Nrf2 in renal epithelial cells co-cultivated with renal fibroblasts, thereby enhancing the expression of cytoprotective TKT. This protective response is not observed in monoculture. Activation of the Nrf2 pathway represents a co-operative cellular strategy of protection against acidosis.

Keywords: Nrf2; cellular crosstalk; chronic kidney diseases; extracellular acidosis; renal cell crosstalk; transketolase.

Figure 1

(a) Clustering of gene expression (https://software.broadinstitute.org/morpheus/; Euclidean distance, accessed on 22 November 2022) according to FPM values. Only genes with FPM > 100 are shown (48 h incubation). Clustering separates the samples clearly according to cell type and pH. (b,c) Overlap of differentially expressed genes by DESeq2 and edgeR analysis in NRK-52E and NRK-49F cells after 48 h exposure to acidic media. (d) Overlap of acidosis-induced differentially expressed genes in NRK-52E and NRK-49F cells (only genes identified by DESeq2 and edgeR were used). (e) Direction of overlapped differentially expressed genes in NRK-52E and NRK-49F.

Figure 2

(a) Differentially expressed genes recognized by IPA with |log2FC| ≥ 1 in NRK-52E and NRK-49F cells. (b) Results of the canonical pathway analysis by IPA for NRK-52E. The Nrf2 pathway has the highest scores. (c) Results of the canonical pathway analysis by IPA for NRK-49F. The Nrf2 pathway has the highest scores.

Figure 3

Results of the upstream regulator analysis by IPA for NRK-52E (left) and NRK-49F (right), applying higher stringency filters. The Nrf2 pathway (NFE2L2) is predicted for NRK-52E but not for NRK-49F cells.

Figure 4

(a,e) Acidosis enhanced Nrf2 mRNA expression in NRK-52E cells in co-culture but not in NRK-49F cells. (n = 4). (b,f) Immunoblots for Nrf2 in three cell fractions of NRK-52E and NRK-49F cells. (c,g) Statistical analysis of Nrf2 expression in the chromatin fraction of NRK-52E (n = 9) and NRK-49F cells (n = 8). (d,h) Exemplary Ponceau-stained membranes (here for immunoblots (b) and (f)) show the protein loading of the gel and successful transfer to the membrane and were used for normalization. (i,j) Nrf2 mRNA expression was not enhanced by acidosis in monoculture (n = 6).

Figure 5

(a–c) Acidosis enhanced pNrf2^SER40 expression to a larger extent than total Nrf2 (dpNrf2/dNrf2) in the chromatin fraction of NRK-52E cells in co-culture. n = 9. * = p < 0.05 versus 1. (e–g) This was not the case for NRK-49F cells. (n = 8). (d,h) Acidosis enhanced the activity of the Nrf2 reporter gene luciferase in NRK-52E cells but not in NRK-49F cells. (n = 10). (b,f) Exemplary Ponceau-stained membranes (here for immunoblots (a,e)) show the protein loading of the gel and successful transfer to the membrane and were used for normalization.

Figure 6

(a–c) Impact of extracellular acidosis on TKT mRNA and protein expression in NRK-52E cells under co-culture conditions. N for mRNA = 4. N for protein = 9. (d–f) Impact of extracellular acidosis on TKT mRNA and protein expression in NRK-52E cells under monoculture conditions. N for mRNA = 4. N for protein = 9. Exemplary Ponceau-stained membranes show the protein loading of the gel and successful transfer to the membrane and were used for normalization.