TSS-Seq analysis of low pH-induced gene expression in intercalated cells in the renal collecting duct

<div><p>Metabolic acidosis often results from chronic kidney disease; in turn, metabolic acidosis accelerates the progression of kidney injury. The mechanisms for how acidosis facilitates kidney injury are not fully understood. To investigate whether low pH directly affects the expression of genes controlling local homeostasis in renal tubules, we performed transcription start site sequencing (TSS-Seq) using IN-IC cells, a cell line derived from rat renal collecting duct intercalated cells, with acid loading for 24 h. Peak calling identified 651 up-regulated and 128 down-regulated TSSs at pH 7.0 compared with those at pH 7.4. Among them, 424 and 38 TSSs were ≥ 1.0 and ≤ -1.0 in Log₂ fold change, which were annotated to 193 up-regulated and 34 down-regulated genes, respectively. We used gene ontology analysis and manual curation to profile the up-regulated genes. The analysis revealed that many up-regulated genes are involved in renal fibrosis, implying potential molecular mechanisms induced by metabolic acidosis. To verify the activity of the ubiquitin-proteasome system (UPS), a candidate pathway activated by acidosis, we examined the expression of proteins from cells treated with a proteasome inhibitor, MG132. The expression of ubiquitinated proteins was greater at pH 7.0 than at pH 7.4, suggesting that low pH activates the UPS. The <i>in vivo</i> study demonstrated that acid loading increased the expression of ubiquitin proteins in the collecting duct cells in mouse kidneys. Motif analysis revealed Egr1, the mRNA expression of which was increased at low pH, as a candidate factor that possibly stimulates gene expression in response to low pH. In conclusion, metabolic acidosis can facilitate renal injury and fibrosis during kidney disease by locally activating various pathways in the renal tubules.</p></div

Immunoperoxidase staining of ubiquitin in the cortex and outer medulla in acid-loaded mouse kidneys.

<p>Mouse kidneys were extracted (A and D) before and (B and E) one and (D and F) three days after HCl loading. The expression of ubiquitin in the cortex and outer medulla was examined. CCD: cortical collecting duct and OMCD: outer medullary collecting duct.</p

TSS-Seq quality.

<p>TSS-Seq quality.</p

Immunoperoxidase staining of H⁺-ATPase B1/2 in the cortex and outer medulla in HCl-loaded mouse kidneys.

<p>Mouse kidneys were extracted (A and D) before and (B and E) one and (D and F) three days after HCl loading. The expression of H⁺-ATPase B1/2 in the cortex and outer medulla was examined. CCD: cortical collecting duct and OMCD: outer medullary collecting duct.</p

Effect of low pH on cell viability.

<p>(A) Cell morphology at pH 7.4 or at pH 7.0 is shown in the upper part. The lower part shows the result of cell viability evaluated by WST-8 assay. (B) Time-dependent change in the expression of H⁺-ATPase B1/2 protein upon exposure to low pH (pH 7.0) evaluated by western blotting.</p

Number of up- or down-regulated TSSs and genes by low pH.

<p>Number of up- or down-regulated TSSs and genes by low pH.</p

Effect of low pH on the ubiquitination of proteins in IN-IC cells.

<p>(A) Representative western blot results for ubiquitinated proteins that were extracted from IN-IC cells incubated at pH 7.4 or at pH 7.0 with MG132. (B) Quantitative analysis of the ubiquitinated proteins by western blot. Band densities higher than 75 kDa were measured. n = 4.</p

GO analysis of genes with expression levels that are up-regulated in pH 7.0 (high stringency, enrichment score > 1.5).

<p>GO analysis of genes with expression levels that are up-regulated in pH 7.0 (high stringency, enrichment score > 1.5).</p

UPS-related genes with expression levels that were up-regulated by low pH.

<p>UPS-related genes with expression levels that were up-regulated by low pH.</p

Stability and reproducibility of the biological duplicates.

<p>(A and C) The correlations of the expression values between duplicates 1 and 2 at pH 7.4 and pH 7.0, respectively. Green dots indicate high reproducibility and red dots indicate low reproducibility. (B and D) The numbers of reproducible or irreproducible TSS clusters between the duplicates. Green and red bars represent the reproducible and the irreproducible clusters, respectively.</p