ATF4 in proximal tubules modulates kidney function and modifies the metabolome

Abstract

Activating transcription factor 4 (ATF4) is a transcription factor that mediates the response to stress at the cellular, tissue, and organism level. We deleted the gene encoding ATF4 in the proximal tubules of the mouse kidney by using a temporal and cell type-specific approach. We show that ATF4 plays a major role in regulating the transcriptome and proteome, which, in turn, influences the metabolome and kidney functions. Genome-wide transcriptomics and single-plot, solid-phase-enhanced sample preparation (SP3)-proteomics studies reveal that ATF4 deletion changes more than 30% of transcripts and, similarly, corresponding proteins in the proximal tubules. Gene Set Enrichment Analysis indicates major changes in transporters, including amino acid transporters. Metabolomic analyses show that these changes in transporters are associated with altered profiles of amino acids in the blood, kidney, and urine. Stable isotope glutamine tracing in primary tubule cells isolated from kidney cortices confirms that ATF4 regulates glutamine transport and metabolism. We suggest that even in the absence of additional stresses, such as kidney injury, ATF4 in the proximal tubules modulates both retention of specific nutrients and excretion of catabolic products like creatinine to maintain normal kidney function. KEY MESSAGES: Activating transcription factor 4 (ATF4) deletion changed more than 30% of genome-wide transcripts and corresponding proteins in the proximal tubules. One set of the profound changes occurred in amino acid transporters and Slc22 family transporters. Changes in transporters were accompanied by altered profiles of amino acids and wastes in the blood, kidney, and urine. ATF4 in the kidney proximal tubules plays a key role in regulating both the reabsorption of nutrients and the excretion of wastes.

Keywords: Activating transcription factor 4 (ATF4); Kidney proximal tubules; Metabolomics; Proteomics; Transcriptomics; Transporters.

Fig. 1

Generation and verification of the ATF4 knockout specifically in proximal tubules. A Generation of GgtCreER; ATF4 knockout (KO) (GCERAΔ) mice. B Genotyping with DNA extracted from the kidney and liver (as negative reference) and PCR as described in the “Methods” section; C Staining of kidney tissues with ATF4 antibody. WT: wild type; GCERA: ggtCreER; ATF4^fl/fl; GCERAΔ: ggtCreER; ATF4^fl/fl after treatment with tamoxifen; K: kidney; L: liver

Fig. 2

Deletion of ATF4 in proximal tubules caused genome-wide transcript changes. A Principal component analysis (PCA) of mRNA-seq data; B volcano plots of -log₁₀(p_adj) versus log₂(fold change) of gene expression in GCERAΔ over WT cortices. p_adj and log₂(fold change) were calculated using R package DESeq2. Significantly changed genes are in blue, purple, and red. Genes significantly downregulated by two-fold are in blue. Genes significantly upregulated by two-fold are in red; C GSEA generated heatmap of top 50 features for each phenotype; D pathways generated by BioJupies; E significantly increased Slc genes by two-fold, and F significantly decreased Slc genes by two-fold

Fig. 3

Deletion of ATF4 in proximal tubules caused proteome wide protein changes. A Principal component analysis (PCA) of proteomics data; B volcano plots of -log₁₀(p_adj) versus log₂(fold change) of protein levels of GCERAΔ over WT. p_adj and log₂(fold change) were calculated using R package DESeq2. Significantly changed proteins are in blue, purple, and red. Proteins significantly downregulated by two-fold are in blue. Proteins significantly upregulated by two-fold are in red; C GSEA generated heatmap of top 50 features for each phenotype; D significantly increased Slc proteins, and E significantly decreased Slc proteins

Fig. 4

Deletion of ATF4 significantly affected amino acid transporters (AATs) and Slc22 family transporters at both mRNA and protein levels. A Venn diagram of all detected transporter mRNAs and proteins; B correlation between shared 123 transporter mRNAs and proteins; C pathways generated by Ingenuity Pathway Analysis (IPA) of all transporter mRNAs. Positive Z score is in red, negative Z-scores are in blue; D significantly increased AATs; E significantly decreased AATs; F significantly increased Slc22 family transporters; G significantly decreased Slc22 family transporters. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.0001

Fig. 5

Deletion of ATF4 in proximal tubules caused changes in metabolite profiles in the serum, kidney, and urine. A, E, I Principal component analysis (PCA) of metabolites in the serum, kidney, and urine; B, F, J volcano plots of -log₁₀(p_adj) versus log₂(fold change) of metabolites of GCERAΔ over WT. Significantly reduced metabolites are in blue; significantly increased metabolites are in red. C, G, K Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of metabolites in the serum, kidney, and urine generated by MetaboAnalyst; D, H, L Small Molecule Pathway database (SMPDB) pathways of metabolites in the serum, kidney, and urine generated by MetaboAnalyst

Fig. 6

Deletion of ATF4 in proximal tubules affects excretion of wastes and reabsorption of nutrients. A–M, metabolites in the serum, kidney, and urine analyzed by metabolomics studies. N–U, markers of renal functions measured by spectrometry. Transporters in red and blue are increased and reduced in GCERAΔ compared to WT, respectively. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.0001

Fig. 7

Deletion of ATF4 in proximal tubules affects glutamine metabolism in primary tubular cells. A, diagram of isolation of primary tubule cells from kidney cortices; B, diagram of uptake and metabolism of [U¹³C₅]glutamine; C–T, abundances of metabolites incorporated or non-incorporated with ¹³C isotopes and their corresponding fractional incorporations at 2 h and 24 h after cells were fed with [U¹³C₅]glutamine. Transporters in blue in B are reduced in GCERAΔ compared to WT. α-KG, α-ketoglutarate. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.0001. Orange stars indicate significance for comparisons of abundances incorporated with ¹³C isotopes between GCERAΔ and WT; black stars indicate significance for comparisons of abundances non-incorporated with ¹³C isotopes between GCERAΔ and WT

Fig. 8

Proposed model of metabolite (mainly amino acids) changes resulted from the expression changes in transporters (mainly amino acid transporters) in kidneys of GCERAΔ mice. Transporters in red and blue are increased and reduced in GCERAΔ compared to WT, respectively