Expression of RhCG, a New Putative NH3/NH4+Transporter, along the Rat Nephron

Abstract

ABSTRACT. Two non-erythroid members of the erythrocyte Rhesus (Rh) protein family, RhBG and RhCG, have been recently cloned in the kidney. These proteins share homologies with specific NH3/NH4+transporters (Mep/Amt) in primitive organisms and plants. When expressed in a Mep-deficient yeast, RhCG can function as a bidirectional NH3/NH4+transporter. The aim of this study was to determine the intrarenal and intracellular location of RhCG in rat kidney. RT-PCR on microdissected rat nephron segments demonstrated expression of mRNAs encoding RhCG in distal convoluted tubules, connecting ducts, and cortical and outer medullary collecting ducts but not in proximal tubules and thick ascending limbs of Henle’s loop. Immunolocalization studies performed on rat kidney sections with rabbit anti-human RhCG 31 to 48 antibody showed labeling of the apical pole of tubular cells within the cortex, the outer medulla, and the upper portion of the inner medulla. All cells within connecting tubules had identical apical staining. In cortical collecting ducts, a subpopulation of cells that has either apical staining (α-intercalated cells) or diffuse staining (β-intercalated cells) for the β1 subunit of the H+-ATPase, was heavily stained at their apical pole with the RhCG antibody while principal cells identified as H+-ATPase negative cells showed a faint apical staining for RhCG that was much less intense than in adjacent intercalated cells. In the outer medulla and the upper portion of the inner medulla, RhCG labeling was restricted to a subpopulation of cells within the collecting duct that apically express the β1 subunit of the H+-ATPase, indicating that RhCG expression in medullary collecting ducts is restricted to intercalated cells. No labeling was seen in glomeruli, proximal tubules, and limbs of Henle’s loop. Immunoblotting of apical membrane fractions from rat kidney cortex with the rabbit anti-human RhCG 31 to 48 antibody revealed a doublet band at approximatively 65 kD.