GATA2 Regulates Body Water Homeostasis through Maintaining Aquaporin 2 Expression in Renal Collecting Ducts

GATA2 is specifically expressed in CD cells of mouse kidney. (A) GFP immunoreactivity (green) in kidney of Gata2^GFP/+ mice is colocalized with DBA-lectin-positive (red) CD cells. (B) Flow cytometry analysis of kidney cells. GFP-positive renal tubular cells (red dots) from Gata2^GFP/+ are sorted and subjected to the qRT-PCR analysis. SSC, side scatter. (C) Transcripts of CD cell-affiliated genes (Ae1, Ae4, and Aqp2) are highly enriched in the GFP-positive renal cells of Gata2^GFP/+ mice in comparison with the whole kidney sample. Expression of proximal or distal tubular markers (NCC, Nkcc2, Sglt1, Sglt2, and Umod) in GFP-positive cells is lower than that in the whole kidney cells. The experiments were repeated three times. Data are shown as the means ± SD. The statistical significance of the differences between the GFP-positive cells and whole kidney cells are indicated (***, P < 0.001; unpaired Student's t test).

Renal tubular cell-specific conditional deletion of Gata2 gene (Gata2-CKO). (A) Experimental time course of doxycycline (dox)-induced tubular cell-specific Gata2 deletion. (B) Cre activity indicated by tdTomato are observed exclusively in the tubular cells (T) but not in the glomeruli (G) of the kidney in the Pax8-rtTA::tetO-Cre::Rosa26TdTomato (R26T) reporter transgenic mice. Scale bars, 100 μm. (C) Schematic diagram of Gata2^GFP, Gata2^flox and recombined Gata2⁻ alleles. Primer pairs used to detect the Gata2^flox allele are depicted. (D) Genomic quantitative PCR analysis of the unrecombined Gata2^flox allele in the collecting duct (CD), spleen (SP), and bone marrow (BM) cells. Data are shown as means ± SD derived from three independent experiments. (E) Gata2 mRNA expression level in the CD, SP, and BM cells of Gata2^flox/GFP control and Gata2-CKO mice. Statistical significance of differences between Gata2^flox/GFP (n = 3) and Gata2-CKO (n = 3) mice is depicted (***, P < 0.001; *, P < 0.05; N.S., not significant).

(A to E) Body weight, blood glucose, serum, and urnie biochemical indexes. Gata2^flox/flox (n = 6) and Gata2-CKO (n = 10) mice showed comparable level of body weight (A), blood glucose level (B), serum level of BUN (blood urea nitrogen) (C), Na⁺, K⁺, and Cl⁻ (D), and total daily urinary excretion of Na⁺, K⁺, and Cl⁻ (E). (N.S., not significant; ***, P < 0.001; Student's unpaired t test).

Renal tubular cell-specific Gata2 deletion leads to high urine volume and low urine osmolality. (A and B) Volume and osmolality of 24-hour urine in Gata2-CKO (n = 10) and littermate control (Gata2^flox/flox; n = 6) mice. Statistical significance of the differences between Gata2-CKO and the control littermates is indicated (***, P < 0.001; Student's unpaired t test). (C) Twenty-four-hour solution intake of control and Gata2-CKO mice (***, P < 0.001; Student's unpaired t test). (D) No obvious histological abnormality in glomeruli (a and b) and CD (c and d) in the Gata2-CKO in comparison with the Gata2^flox/GFP littermate control mice. Scale bars, 50 μm.

Gene expression profile in CD cells. (A) Heat map generated from DNA microarray data of water reabsorption-related genes changed in CD cells of Gata2 CKO mice. Heat map colors indicate normalized expression level (log₂). Genes are categorized into three groups by their function using Ingenuity pathway analysis (IPA) software and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg/pathway.html). (B) mRNA expression of Aqp3, Aqp4, Gata3, Creb, and Avpr2 in the CD cells of Gata2^flox/flox and Gata2-CKO mice examined by qRT-PCR analysis. The statistical significance of the difference between the Gata2^flox/flox and Gata2 CKO mice is indicated (*, P < 0.05; Student's unpaired t test, N.S; not significant).

GATA2 is important for maintenance of Aqp2 expression. (A) Aqp2 mRNA level is decreased in the DBA-sorted CD cells of Gata2-CKO mice (n = 3) compared to that of the Gata2^flox/GFP control mice (n = 3). (B) Representative immunoblot analysis of Aqp2 using whole-cell extract of kidney in Gata2^flox/flox control (n = 3) and Gata2-CKO (n = 3) mice. (C) Aqp2 protein level is quantified and normalized to the α-tubulin level. Data are presented as the means ± SD. The statistical significance of the differences between Gata2 CKO and the Gata2^flox/flox control littermates is indicated (*, P < 0.05; Student's unpaired t test). (D) Colocalization of GFP and Aqp2 immunoreactivities in renal tubules of the Gata2^flox/GFP mouse. (E) Immunofluorescence analysis shows the reduced Aqp2 expression in the outer and inner medulla of kidney in the Gata2-CKO mouse. Scale bar, 50 μm.

GATA2 is more crucial than GATA3 for Aqp2 trans activation in vivo. (A) The stacked bar graph shows the relative abundances of Gata2 and Gata3 transcripts in the Gata2^flox/flox (n = 3) and Gata2 CKO (n = 4) mice. The statistically significant changes in the Gata2 CKO mice compared with the Gata2^flox/flox are indicated (*, P < 0.05; Student's unpaired t test). Gata2^GFP/+ but not Gata3^LacZ/+ mice show increase in urine volume (B) and decrease in Aqp2 mRNA level (C). Statistical significance of differences is indicated (*, P < 0.05; Student's unpaired t test).

GATA2 directly activates Aqp2 expression through the evolutionary conserved GATA sites in the promoter region. (A) BirA/FLBio system for chromatin pulldown assay. Flag and biotinylation tags are fused to the N terminus of GATA2 protein. FLBio-GATA2 and E. coli BirA biotin ligase are stably cotransfected into mIMCD cells. (B) Confirmation of FLBio-GATA2 expression in the BirA/FLBio GATA2 mIMCD cells by immunoblotting analysis. The mIMCD cell line transfected only with BirA is used for control. Immunoblotting using GATA2 antibody detected the FLBio-GATA2 and endogenous GATA2 proteins in the FLBio-GATA2-transfected cells, while the BirA-only transfected cells express only the endogenous GATA2 protein (top panel). Biotinylated GATA2 is detected by streptavidin-HRP and is absent from the BirA-only transfected cells (middle panel). Lamin B antibody is used as loading control (bottom panel). (C) Chromatin pulldown experiments show that FLBio-GATA2 binds to the Aqp2 promoter in the mIMCD cell. Two genomic DNA regions around Gata1 locus (G1-3.8 kb and G1-14.4 kb) are used as negative controls. Data are presented as means ± SD from three independent experiments. Statistical significance of differences is indicated (*, P < 0.05; Student's unpaired t test). (D) Construction of 1.5-kb wild-type (Aqp2-WT-LUC) or GATA site-mutated (Aqp2-mut-LUC) Aqp2 promoter-luciferase reporter. Asterisks indicate nucleotides that are conserved between human and mouse Aqp2 genes. (E) Cotransfection of GATA2-expressing plasmid (pEF-GATA2) in the mIMCD cells activates the Aqp2-WT-LUC reporter but not the Aqp2-WT-LUC reporter. Data are presented as means ± SD from three independent experiments. Statistical significance of differences is indicated (***, P < 0.001; Student's unpaired t test).