Understanding the molecular basis of the complex regulatory networks controlling renal ion transports is of major physiological and clinical importance. In this study, we aimed to identify evolutionarily conserved critical players in the function of the renal distal convoluted tubule (DCT) by a comparative transcriptomic approach. We generated a transgenic zebrafish line with expression of the red fluorescent mCherry protein under the control of the zebrafish DCT-specific promoter of the thiazide-sensitive NaCl cotransporter (NCC). The mCherry expression was then used to isolate from the zebrafish mesonephric kidneys the distal late (DL) segments, the equivalent of the mammalian DCT, for subsequent RNA-seq analysis. We next compared this zebrafish DL transcriptome to the previously established mouse DCT transcriptome and identified a subset of gene products significantly enriched in both the teleost DL and the mammalian DCT, including SLCs and nuclear transcription factors. Surprisingly, several of the previously described regulators of NCC (e.g., SPAK, KLHL3, ppp1r1a) in the mouse were not found enriched in the zebrafish DL. Nevertheless, the zebrafish DL expressed enriched levels of related homologues. Functional knockdown of one of these genes, ppp1r1b, reduced the phosphorylation of NCC in the zebrafish pronephros, similar to what was seen previously in knockout mice for its homologue, Ppp1r1a. The present work is the first report on global gene expression profiling in a specific nephron portion of the zebrafish kidney, an increasingly used model system for kidney research. Our study suggests that comparative analysis of gene expression between phylogenetically distant species may be an effective approach to identify novel regulators of renal function.
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The authors would like to thank Drs. Jelena Kühn-Georgijevic and Lennart Opitz (Functional Genomics Center Zurich) for their assistance with RNA-Seq and bioinformatics analysis. Valuable bioinformatics support was also provided by Ville Kytölä (Genevia Technologies Ltd). We would also like to thank Kerstin Dannenhauer and Michèle Heidemeyer for their technical support including the excellent care of the zebrafish and the careful execution of the HEK293 cell experiments. This work was supported by the Hartmann Müller Stiftung (to YS), the Forschungskredit from the Faculty of Medicine at the University of Zurich (to YS), the RiMED Foundation (to CCC), the Zurich Center for Integrative Human Physiology (to SN, JL), by project grants (310030_143929/1, 310030_173276) from the Swiss National Science Foundation (to JL) and the Swiss National Center for Competence in Research “Kidney.CH” (to JL).
Conflict of interest
The authors declare that they have no conflict of interest.
This article is part of the special issue on Functional Anatomy of the Kidney in Health and Disease in Pflügers Archiv—European Journal of Physiology.
Electronic supplementary material
Raw read count and gene expression table. Gene expressions were quantified from alignment files using htseq-count (version 0.6.0.). Raw counts are reported in this table in columns C–H. R software (version 3.3.1) was used to process the raw counts to FPKM normalized gene expressions. R libraries biomaRt (2.30.0) and GenomicFeatures (1.26.4) were used to annotate Ensembl gene IDs with gene symbols and extract exon lengths for the normalization. FPKM (fragments per kilobase of transcript per million mapped reads) expressions are reported in columns J–O. Library sizes for the FPKM normalization were calculated as a sum of all gene-wise read counts in each sample. Ribosomal RNA-coding genes were removed prior to library size estimation to remove bias caused by rRNA derived reads. (XLSX 4157 kb)
Differential expression between DL segment and whole kidney samples. Differential expression was calculated in R software using library DESeq2 (1.14.1). Table column base Mean describes the average expression across all samples in natural (non-log2) scale. Column “log2FoldChange” shows log2 ratio of expressions between DL segment and whole kidney sample groups with positive values indicating higher expression in DL segment group and vice versa. Remaining columns show error estimates, t test statistics and univariate and multiple testing adjusted p values for t testing difference in group means. (XLSX 3075 kb)
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Sugano, Y., Cianciolo Cosentino, C., Loffing-Cueni, D. et al. Comparative transcriptomic analysis identifies evolutionarily conserved gene products in the vertebrate renal distal convoluted tubule. Pflugers Arch - Eur J Physiol 469, 859–867 (2017). https://doi.org/10.1007/s00424-017-2009-8
- Distal convoluted tubule
- NaCl cotransporter
- Comparative transcriptomics