Single-tubule RNA-Seq uncovers signaling mechanisms that defend against hyponatremia in SIADH

Abstract

In the syndrome of inappropriate antidiuretic hormone secretion (SIADH), hyponatremia is limited by onset of vasopressin-escape caused by loss of the water channel aquaporin-2 in the renal collecting duct despite high circulating vasopressin. Here, we use the methods of systems biology in a well-established rat model of SIADH to identify signaling pathways activated at the onset of vasopressin-escape. Using single-tubule RNA-Seq, full transcriptomes were determined in microdissected cortical collecting ducts of vasopressin-treated rats at 1, 2, and 4 days after initiation of oral water loading in comparison to time-control rats without water loading. The time-dependent mRNA abundance changes were mapped to gene sets associated with curated canonical signaling pathways and revealed evidence of perturbation of transforming growth factor β signaling and epithelial-to-mesenchymal transition on Day 1 of water loading simultaneous with the initial fall in Aqp2 gene expression. On Day 2 of water loading, transcriptomic changes mapped to Notch signaling and the transition from G0 into the cell cycle but arrest at the G2/M stage. There was no evidence of cell proliferation or altered principal or intercalated cell numbers. Exposure of vasopressin-treated cultured mpkCCD cells to transforming growth factor β resulted in a virtually complete loss of aquaporin-2. Thus, there is a partial epithelial-to-mesenchymal transition during vasopressin escape with a subsequent shift from quiescence into the cell cycle with eventual arrest and loss of aquaporin-2.

Keywords: TGFβ; aquaporin-2; cell cycle; cortical collecting duct; transcriptome.

Figure 1. Experimental model

A. The study design was adapted from Ecelbarger et al.. Rats were euthanized on day 1, 2, and 4. B. Immunoblotting for AQP2 in the cortex, outer medulla, and inner medulla (n=4 per group) at day 2 of escape protocol. C. Densitometry shows a significant decrease in aquaporin-2 protein abundance in the cortex and inner medulla of the escape animals, consistent with previous reports. For each lane, 10 g (cortex), 5 μg (outer medulla), and 2 μg (inner medulla) of total protein was loaded. *p < 0.05 by unpaired t-test. (Coomassie-stained gels, run in parallel, showed equal loading.) Glyc-AQP2, glycosylated aquaporin-2; non-Glyc-AQP2, nonglycosylated aquaporin-2.

Figure 2

Distribution of Z* values for Log₂(Escape/Control) across all genes. Z* is estimated as mean value for all replicates divided by the standard deviation across means for all genes.

Figure 3

Abundances of aquaporin-2 (AQP2) and aquaporin-3 (AQP3) transcripts in the CCD are decreased in vasopressin-escape animals. A. RNA-Seq reads are mapped to the RefSeq transcript models of for rat Aqp2 gene (top) and Aqp3 gene (bottom). For each RefSeq transcript, exons are shown as red rectangles and introns as blue lines connecting the exons. Barbs indicate direction of transcription. The coverage was calculated as the number of reads per million nucleotides in the whole genome. B. Normalized RNA-Seq read counts for Aqp2 and Aqp3 in the CCD segments obtained from four pairs (control versus escape animals) at day 2. C. Change in AQP2 and AQP3 transcript abundances in the microdissected CCDs on different days relative to time controls (*Padj < 0.05 for escape vs control at each time point, paired t-test).

Figure 4

Changes in transcript abundances for TF genes associated with annotated KEGG pathways during the onset of vasopressin-escape.

Figure 5

Official gene symbols of transcripts associated with TGFβ/activin/BMP signaling undergoing changes during onset of vasopressin-escape. Increased, green; decreased, red. See Table 4 for annotations of these genes. Specific values are available in Supplemental Dataset 2.

Figure 6. Onset of vasopressin-escape is associated with a partial epithelial-to-mesenchymal transition in rat CCD cells

A. Time course of transcript abundance changes for selected EMT marker genes during onset of vasopressin-escape in microdissected rat CCDs shows increase in mesenchyme-associated transcripts (Col1a1, Col6a3, Mmp2 and Vim) without loss of epithelium-associated transcripts (Cdh1 and Ocln). Asterisk indicates Benjamini-Hochberg FDR-adjusted P value <0.05 (see Supplementary Table 2 for standard errors). B. Immunocytochemical labeling for AQP2 and AQP3 in renal cortex of rats undergoing vasopressin-escape shows retention of normal epithelial polarity. VDAC labeling of mitochondria was also carried out to reveal presence of intercalated (‘mitochondria-rich’) cells. DAPI labeling of nuclei facilitates recognition of apical versus basal aspects of cells.

Figure 7. TGFβ exposure decreases AQP2 protein and mRNA abundance in mpkCCD cells

A. Representative immunoblot showing relative AQP2 abundances in control (CTR) cells and cells exposed to TGFβ (1, 5 or 10 ng/ml) for 2 days in serum-free medium. The cells were pretreated with 1 nM dDAVP on the basolateral side for 4 days to induce AQP2 expression. Bottom panel shows a Coomassie-stained gel to demonstrate equal loading. B. AQP2 band density was significantly decreased both for glycosylated and nonglycosylated AQP2 (n=3). C. SYBR Green™ fluorescence curves for RT-qPCR experiments quantifying AQP2 mRNA under the control (CTR) condition, after 1 ng/ml TGFβ (2 days) and after 1 ng/ml TGFβ plus amiloride at 10 μM (2 days). Cells were pre-treated with dDAVP at 1 nM for 4 days. Horizontal green line is the threshold used to calculate C_t. The relative AQP2 abundances (normalized to the housekeeping gene RPLP0) are shown as a bar graph.

Figure 8. Cell counting in immunofluorescently labeled CCDs microdissected from rats

After microdissection, tubules were fixed with paraformaldehyde followed by immunofluorescence staining for intercalated cells (using pendrin and H+-ATPase antibodies) and nuclei (DAPI). A. Standard confocal fluorescence image of a microdissected CCD from a control vasopressin-treated rat. Pendrin, green; B1 H-ATPase, red; DAPI labeling of nuclei, blue. Examples of identified α-intercalated cells (α-IC) and β-intercalated cells (β-IC) are pointed out. Asterisks indicate cells that appear to lack pendrin labeling in confocal image but are revealed to be β-intercalated cells in the 3D reconstruction. B. Maximum intensity projection of 3-D reconstructed tubule image generated by IMARIS image analysis software. Colors are the same as in Panel A. Asterisks mark the same cells indicated in Panel A. C. IMARIS-generated analytical image. The pendrin-positive IC cells and non-pendrin IC cells were labeled in green and red, respectively, based on IMARIS surface and spot analysis tools. Nuclei are shown in cyan. For details, see Supplemental Figure 2.

Figure 9

Transcripts corresponding to most genes present in the KEGG Cell Cycle Pathway were increased in abundance in CCDs from rats on Day 2 of development of vasopressin-escape. Values for all four Day-2 replicates are shown as individual blocks colored to show magnitude of change (red, increased; green, decreased) as generated by Bioconductor Pathview software (https://www.bioconductor.org/packages/devel/bioc/html/pathview.html) run on R. The specific values are available in Supplementary Dataset 1.

Figure 10

Time courses of abundance change for mRNAs coding for protein kinases (left) and TFs (right) involved in regulation of cell-cycle. All six cell-cycle related transcripts (black) showed significant increases only on Days 2 and 4. Only Foxm1 was significantly changed on Day 1, a decrease. Abundant non-cell-cycle transcripts (Prkaca and Hoxb7), shown in red, are included as controls. Cdk1, Cyclin-dependent kinase 1; Plk1, Polo-like kinase 1; Aurkb, Aurora B kinase, Prkaca, Protein kinase A catalytic α; E2f1, E2F TF 1; Timeless, timeless circadian clock; Foxm1, Forkhead Box M1; Hoxb7, Homeobox B7.

Figure 11. Cell cycle indices in renal CCDs from vasopressin-escape versus control rats

A. Nuclei labeled with antibody to PCNA (S-phase marker) are scarce. Image shows section of renal cortex from rat undergoing vasopressin-escape (Day 2). Labeling for AQP2 (green) and the B1 subunit of the vacuolar proton-ATPase (red) identify collecting ducts (numbered). A single collecting duct cell with PCNA labeling (cyan) is seen (red arrow). DAPI, blue. B. Distribution of integrated DAPI fluorescence signal in nuclei of microdissected CCDs from rats undergoing vasopressin-escape (Day 4 of escape protocol) versus control rats. See Table 8 for quantitation.

Figure 12. AQP2 protein is strongly expressed in cultured CCD cells in G0, but not when in the cell cycle

dDAVP-treated (1 nM) mpkCCD cells were grown on a semi-permeable filter and synchronized in the different phases of the cell cycle (FCS withdrawal for G0 phase, PD0332991 for G1, Hydroxyurea for S and Nocodazole for G2/M phase), then immunoblotted for AQP2 and Cyclins D1, A, and B.