Expression of acid base transporters in the kidney collecting duct in Slc2a7 -/-

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1 Supplemental Material Results. Expression of acid base transporters in the kidney collecting duct in Slc2a7 -/- and Slc2a7 -/- mice. The expression of AE1 in the kidney was examined in Slc26a7 KO mice. As indicated in Fig 2, A, Supplement, AE1 mrna expression increased in the outer medulla of Slc2a7 -/- mice relative to Slc2a7 -/- mice (P<0.05, n=5). Immunofluorescence labeling (Fig. 2, B, Supplement) and Western blotting (not shown) indicate increased abundance of AE1 protein in OMCD of Slc2a7 -/- ko mice. Figure 2, C demonstrates the reduction in the expression of pendrin mrna in Slc26a7 ko mice relative to wild type littermates. Expression of gastric H-K-ATPase and AE2 in the stomach. Double immunofluorescence labeling was performed on Slc26a7 wt and ko stomachs. Figure 3, A, top column (Supplement) demonstrates the expression of AE2 (left panel) and gastric H-K-ATPase (right panel), with the middle panel depicting the merged image in wild type (Slc26a7 +/+ ) mouse stomach. Figure 3, A, bottom column (Supplement) demonstrate the expression of AE2 (left panel) and gastric H-K-ATPase (right panel), along with the merged image (middle panel) in Slc26a7-null mouse. The results indicated normal abundance and localization of AE2 and H-K ATPase in gastric parietal cells in Slc26a7 -/- mice relative to Slc26a7 +/+ mice. Western blotting of gastric H-K- ATPase on membrane proteins isolated from corpus of the stomach and showed no difference in the abundance of gastric H-K-ATPase in adult Slc26a7 -/- mice (data not shown). This is very distinct from Slc26a9, another member of Slc26 anion transporter family, which is expressed in tubulovesicles of gastric parietal cells and its deletion

2 causes loss of secretory canaliculi, reduction in gastric parietal cells and complete absence of acid secretion (10). Microscopic analysis of stomach and kidney in Slc26a7-null mice. Microscopic analysis of H and E prepared sections showed gastric glands in 6-7 weeks old Slc26a7-/- mutant mouse to be normal (Fig. 4, Top, lower panels, a and b; Supplement) relative to Slc26a7+/+ mouse (Fig. 4, Top, upper panels, a and b; Supplement). Parietal cells (arrow) with their lighter cytoplasm and distinct organization were present at normal numbers in Slc26a7-/- mice (Fig. 4, Supplement). Zymogen cells were similarly normal in Slc26a7 mutant stomach. The cell area and nuclear area of parietal cells from the Slc26a7 +/+ and Slc26a7 +/- were not significantly different. H and E analysis showed normal kidney structures in Slc26a7 ko mice, including proximal tubule, thick limb, distal tubule and collecting ducts (Fig. 4, Bottom) (Supplement). Methods. Histology of stomach and kidney. Several pairs of age-matched littermates were analyzed by light microscopy. Mice were 6-7 weeks old at termination and included male and females. Five µm thick sections were stained with hematoxylin and eosin (H&E), or with periodic-acid Schiff (PAS) and alcian blue (AB) for light microscopy. Morphometric analysis of the glandular and forestomach epithelium was performed as previously described (10). The percentage of cell types in a gastric gland was derived from cell counts of randomly selected but well oriented gastric glands beginning at the base and continuing to

3 the lumen of the stomach. Parietal cells were required to contain a nuclear profile, predominant large and well organized mitochondrial distribution and visible lucent areas of smooth membranes (canaliculi and tubulovesicles). Zymogenic cells were identified as having a profile of nucleus, basophilic cytoplasm (RER), and a minimum of three birefringent granules. Mucus neck cells were dark and small, mucus pit cells were determined by their mucus granules. RNA isolation and Northern blot hybridization. Total cellular RNA was extracted from stomach and cortex and medulla of kidney, according to established methods. Hybridization was performed according to established protocols (38). Kidney bots were examined for the expression of Slc26a7, AE1, pendrin and AQP2. Stomach blots were analyzed for the expression of mrnas encoding gastric H +,K + -ATPase - subunit, AE2, and Slc26a7 using 32 P-labeled cdna probes. The membranes were washed, blotted dry, and exposed to a Phosphorimager screen (Molecular Dynamics, Sunnyvale, CA). The densities of each band were normalized to that of the 18S rrna band (as a loading control). Immunofluorescent labeling in mouse stomach and kidney. Single and double immunofluorescent labeling on frozen sections from stomachs or kidneys was performed as described using AE2, AE1, Slc26a7, or V-ATPase polyclonal or gastric H-K-ATPase (beta subunit) monoclonal antibodies as described (28, 29). Western blotting of gastric H-K- ATPase in the stomach of Slc26a7 +/+ and Slc26a7 -/- mice. The corpus mucosa was dissected with a razor blade, washed, and centrifuged. Protein extracts were prepared as described (10) in the presence of a cocktail of protease inhibitors (Complete; Roche Diagnostics, Mannheim, Germany). Western

4 blotting was performed as before using H-K ATPase antibodies. Measurement of Gastric ph and Acid/Base Equivalents. The ph and acid-base content of gastric secretions of Slc26a7 / and wild-type littermates were measured at days of age as described previously (10, 13). Mice were fasted overnight, euthanized 15 min after subcutaneous injection of histamine (2 µg/g body weight) and the intact stomachs were removed. The gastric contents, which would include both basal and histamine-stimulated acid-base equivalents, were rinsed into 5 ml of oxygen-saturated normal saline solution at room temperature, degassed, pelleted by centrifugation, and the ph and acid-base equivalents were determined by titration with NaOH. Materials- 32 P-dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). RadPrime DNA labeling kit was purchased from Gibco BRL, USA. Statistical Analyses. Data for microcopic and morphometry were analyzed using SigmaPlot, and means and standard errors, and unpaired t-tests were determined by genotype and gender. Results were considered significant when p < Differences in gastric ph and acid secretion in the stomach in wild type and null mice were evaluated statistically by ANOVA (Mann-Whitney test). Figure legends. Fig. 1. Expression of Slc26a7 in the kidney. Double immunofluorescent labeling of AQP2 and Slc26a7 demonstrate the expression of Slc26a7 on the basolateral membrane of A-intercaleted cells in the OMCD. The principal cells are delineated by their apical

5 labeling for AQP2. Arrows in the merged image point to the basolateral labeling by Slc26a7 and the apical labeling by AQP2. Fig. 2. Expression of acid base transporters in the kidney of Slc26a7+/+ and Slc26a7-/- mice. A. Northern hybridization of AE1 in the outer medulla. B. Immunofluorescence labeling of AE1 in the outer medulla. C. Expression of pendrin in the cortex. Fig. 3. Double immunofluorescent labeling of AE2 and H-K-ATPase. Top column demonstrates the expression of AE2 (left panel) and gastric H-K- ATPase (right panel) in wild type mouse stomach. The middle panel is a merged image. Bottom column demonstrates the expression of AE2 (left panel) and gastric H-K- ATPase (right panel), along with the merged image (middle panel), in Slc26a7-null mouse. Figure 4. Histopathologic analysis of stomach and kidney in Slc26a7 null mice. A. H and E analysis of stomach sections in Slc26a7-/- and Slc26a7+/+ mice. Parietal cells (arrows) had normal abundance and appearance in Slc26a7-/- mice (A, Bottom) relative to Slc26a7+/+ (A, Top). Zymogen cells and mucous cells in Slc26a7-/- stomach appeared normal. B. H and E analysis of kidney sections in Slc26a7-/- and Slc26a7+/+ mice. Kidney tubules and cells in the proximal and distal tubules had normal appearance and morphology in Slc26a7-/- mice (B, Bottom) relative to Slc26a7+/+ (B, Top).

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9 Figure 4. Histopathologic analysis of stomach (A) and kidney (B) in Slc26a7 null mice.

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