Distal renal tubular acidosis in mice that lack the forkhead transcription factor Foxi1

Abstract

While macro- and microscopic kidney development appear to proceed normally in mice that lack Foxi1, electron microscopy reveals an altered ultrastructure of cells lining the distal nephron. Northern blot analyses, cRNA in situ hybridizations, and immunohistochemistry demonstrate a complete loss of expression of several anion transporters, proton pumps, and anion exchange proteins expressed by intercalated cells of the collecting ducts, many of which have been implicated in hereditary forms of distal renal tubular acidosis (dRTA). In Foxi1-null mutants the normal epithelium with its two major cell types - principal and intercalated cells - has been replaced by a single cell type positive for both principal and intercalated cell markers. To test the functional consequences of these alterations, Foxi1(-/-) mice were compared with WT littermates in their response to an acidic load. This revealed an inability to acidify the urine as well as a lowered systemic buffer capacity and overt acidosis in null mutants. Thus, Foxi1(-/-) mice seem to develop dRTA due to altered cellular composition of the distal nephron epithelium, thereby denying this epithelium the proper gene expression pattern needed for maintaining adequate acid-base homeostasis.

Figure 1

TEM of kidney CCD cells in WT (A and C) and Foxi1^–/– (B and D) mice. Red arrows mark cells with a protruding “tussock-like” apex that are rich in mitochondria (A and C). Cells with this appearance are missing from CCD of Foxi1^–/– origin (B and D). Scale bars: A and B, 5 ∝m; C and D, 2 ∝m.

Figure 2

The cRNA in situ hybridization of mouse kidney sections of WT and Foxi1^–/– origin using antisense probes for Foxi1 (A–D) and Pds (E–G). In cortical sections, Foxi1-positive cells are localized to tubular regions (A). At higher magnification, a scattered pattern of hybridization signals is clearly visualized. While some cells are densely stained, others remain unstained (B). In medulla sections, cells positive for Foxi1 are scattered and distributed along the collecting ducts (C). At a higher magnification, it is evident that some cells stain and others lack any clear hybridization signal (D). The antisense Pds cRNA probe identifies a pattern of scattered tubular cells in cortical sections (E–G). No signals from cells in the medulla can be identified (E). At higher magnification, it is clear that some cells stain, while others do not (F and G), a pattern very similar to that for Foxi1 in the cortex (A and B). No Pds signals can be identified in Foxi1^–/– kidneys. Scale bars: 40 ∝m.

Figure 3

Confocal images of cortical sections using Ab’s against Pds (green) (A–C), AE1 (yellow) (D–G) as well as ToPro3 for nuclear staining (red). In WT kidneys, an apical/luminal staining pattern is visualized, which agrees well with previous observations (12). Apical Pds signals mark β-intercalated cells (12). In Foxi1^–/– sections, no Pds signal can be detected. In the cortex of WT mice, tubular cells are AE1 positive (D), mostly at their basolateral border (E). In the medulla, a similar pattern is visualized (F and G). The lumen is marked with a dashed white line in F. In Foxi1^–/– kidneys, no parenchymal staining can be identified; only erythrocytes stain (D–E), since the antiserum (apart from identifying the kidney and α-intercalated cell-specific isoform of AE1) also recognizes the erythrocyte isoform of AE1 (41). Scale bars: 10 ∝m.

Figure 4

In confocal images of WT kidney sections, β-intercalated cells stain positive for Pds (green) and AE4 (blue) at their apical and basolateral border, respectively. Principal cells stain positive for AQP2 (orange) at their apical aspect (A). While AE4 staining is absent in Foxi1^–/– kidneys (B) (lumen marked with a dashed white line), AQP2 signals are present in both WT and Foxi1^–/– sections (C). AQP2 appears to stain a high fraction of epithelial cells in Foxi1^–/– sections (right panel), as compared with WT (left panel) (C). ATP6B1 is absent from distal nephron epithelia in Foxi1^–/– mice both in cortex (right panels) (D and E) and medulla (right panels) (F and G), as compared with WT (left panels) (D–G). Scale bars: 10 ∝m.

Figure 5

Transcript levels and transcriptional regulation in WT and Foxi1^–/–. Northern blot analysis of whole-kidney RNA preparations derived from either WT or Foxi1^–/– kidneys (A). While Foxi1, Pds, and AE1 signals are absent in kidney RNA prepared from Foxi1^–/– mice, the same levels of ATP6B1 and Kcc4 signals are present in WT and Foxi1^–/– RNA preparations. At least three independent animals (for each genotype) were examined for each probe in three independent experiments, with representative results shown. Results shown in A represent RNA from different mice. Transfection assays (B), promoter reporter gene constructs for AE1, and Pds, were transfected to COS-7 cells, together with an FOXI1 expression vector (20 and 40 ng) with or without insert. Reporter gene activity is shown as fold induction relative to an expression vector void of FOXI1 insert.

Figure 6

Distribution and identity of Foxi1 positive cells. Confocal images of cortical tubules (A). Left image is an overview, right image is a close up of a typical tubule, lumen (L). Pds (green) stains the apical region of β-intercalated cells (white arrows); AE1 (red) stains the basolateral region of α-intercalated cells (blue arrows). Due to presence of overlapping specificity, erythrocytes stain red in a scattered pattern (see below). (B) In the left image, a Foxi1-specific cRNA probe stains tubular cells dark blue, marked with blue and red arrows. The right image shows a confocal image of the left panel using a Pds-specific Ab for immunohistochemistry. While apical/luminal regions of some tubular cells are Foxi1 positive and also stain positive for Pds (red arrows), other such cells are negative for Pds (blue arrows). (C) In the left image, a Foxi1-specific cRNA probe stains tubular cells dark blue (marked with blue and red arrows). The right image shows a confocal image of the left panel using a AE1-specific Ab for immunohistochemistry. While the basolateral region of a tubular cell that is Foxi1 positive also stains positive for AE1 (red arrow), other such cells are negative for AE1 (blue arrows). In right panel of C, several erythrocytes stain (clusters marked with white arrowheads), since the antiserum used (apart from detecting the kidney isoform of AE1) also identifies the erythrocyte isoform of AE1 (see corresponding section in Results). Foxi1 cRNA probes were used to identify Foxi1-positive cells in the cortex and medulla tubuli (left panels) (D and E). A scattered pattern of Foxi1-positive cells can be visualized (red arrows). These sections were then subjected to immunohistochemistry using a AQP2-specific Ab (green, right panels). (D and E) Nuclei were stained red with ToPro3. When Foxi1-positive cells (red and blue arrows) are compared with AQP2-positive cells (green), there is no overlap between these two markers. Thus, Foxi1 most likely is expressed in intercalated cells (B and C), while principal cells stain negative for Foxi1 (D and E). Scale bars: 10 ∝m.

Figure 7

Response to an acute acidic load (see Methods). WT (pH: 1.01 ± 0.26) mice are capable of acidifying their urine significantly more (P < 0.02, n = 5) than Foxi1^–/– (pH: 0.31 ± 0.33) mice 4 hours after this challenge (A). Twenty-four hours after this load (B), WT mice had normal base excess values (–0.08 ± 1.18), whereas Foxi1^–/– mice had consumed more of their systemic buffering capacity (–5.00 ± 0.55; P < 0.002, n = 4). BE, base excess values.

Figure 8

Cellular distribution of intercalated and principal cell markers. Confocal analysis of sections from cortex and medulla of WT and Foxi1^–/– origin (A). Sections were stained using specific antiseras against CAII (intercalated cell marker, blue) and AQP2 (principal cell marker, green); nuclei were stained with ToPro3 (red). When overlays are compared in WT sections, specific subsets of cells stain positive for either CAII (intercalated cells) or AQP2 (principal cells). On the other hand, in Foxi1^–/– sections the two signals overlap completely, and one cell population is identified, positive for both markers. While the AQP2 signal in WT sections is localized to the apical aspect of principal cells, the AQP2 signal is more diffuse and generalized in Foxi1^–/– epithelia. When relative cell numbers are compared (B), it is obvious that only one cell population positive for both markers exists in Foxi1^–/– epithelia. C, cortex; OM, outer medulla; IM, inner medulla. Scale bars: 10 ∝m.

Figure 9

A schematic view of a hypothetical differentiation scheme for principal and intercalated cells. Embryonic precursor cells of ureteric bud origin gradually acquire an epithelial phenotype. These cells are positive for both CAII and AQP2. In the next, Foxi1-regulated step, two separate cell populations arise: principal cells (Foxi1^–, CAII^–, AQP2⁺) and intercalated cells (Foxi1⁺, CAII⁺, AQP2^–).