Figure 7. In vivo analyses of IRR function.

(A) Rats were anesthetized, and their kidneys were perfused through renal arteries with 1% solution of NaHCO₃ or with 0.9% NaCl (control). The kidneys were then removed and immediately homogenized and extracted, as described in Experimental Procedures. The extracts were immunoprecipitated with affinity purified anti-rat IRR antibody or with the same concentration of rat IgGs as control and analyzed by Western blotting with anti-pY antibody. The blot is representative of three independent experiments. The arrow indicates position of the IRR β-subunit band. (B) Blood gas analyses in IRR^+/+ and IRR^−/− mice before and after 7 days of alkali-load administered as 0.28M solution of NaHCO₃ in the drinking water. (C) Initial renal response to alkali-loading measured in IRR^+/+ and IRR^−/− mice. Six hours urine collections (from 9h00 a.m. to 3h00 p.m.) were obtained from same IRR^+/+ and IRR^−/− mice administrated either dionized water on the first day of the experiment (black bars) or NaHCO₃ solution on the second day (white bars) to measure urine pH and renal bicarbonate excretion. (D) Immunoblots of membrane fractions from the renal cortex obtained from IRR^+/+and IRR^−/− mice after 7 days of alkali-loading. The membrane preparations were blotted with antibodies directed against Pds and Atp6v1b1. Densitometric analyses of data show the abundance of Pds, and Atp6v1b1, respectively, expressed as % of control. Equality of loading was verified by running, in parallel, gels that were stained with Coomassie blue and quantified as described previously (Quentin et al., 2004a). Values are means ± SE. See also Figure S6.