Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis

Abstract

Proximal renal tubular acidosis associated with ocular abnormalities such as band keratopathy, glaucoma, and cataracts is caused by mutations in the Na(+)-HCO(3)(-) cotransporter (NBC-1). However, the mechanism by which NBC-1 inactivation leads to such ocular abnormalities remains to be elucidated. By immunological analysis of human and rat eyes, we demonstrate that both kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters are present in the corneal endothelium, trabecular meshwork, ciliary epithelium, and lens epithelium. In the human lens epithelial (HLE) cells, RT-PCR detected mRNAs of both kNBC-1 and pNBC-1. Although a Na(+)-HCO(3)-cotransport activity has not been detected in mammalian lens epithelia, cell pH (pH(i)) measurements revealed the presence of Cl(-)-independent, electrogenic Na(+)-HCO(3)-cotransport activity in HLE cells. In addition, up to 80% of amiloride-insensitive pH(i) recovery from acid load in the presence of HCO(3)(-)/CO(2) was inhibited by adenovirus-mediated transfer of a specific hammerhead ribozyme against NBC-1, consistent with a major role of NBC-1 in overall HCO(3)-transport by the lens epithelium. These results indicate that the normal transport activity of NBC-1 is indispensable not only for the maintenance of corneal and lenticular transparency but also for the regulation of aqueous humor outflow.

Figure 1

Characterization of anti–NBC-1 Ab’s. Lane 1: nontransfected ECV304 cells. Lane 2: cells transfected with kNBC-1 cDNA. Lane 3: cells transfected with pNBC-1 cDNA. Western blot analysis was performed using the Ab’s against the NBC-1 C terminus (common), the kNBC-1 N terminus (k-specific), and the pNBC-1 N terminus (p-specific). A representative blot from three independent experiments is shown.

Figure 2

Immunolocalization of NBC-1 in the eyes. (a) Peroxidase-antiperoxidase staining of the human eye in the absence, Ag(-), and presence, Ag(+), of the antigen peptide. NBC-1 staining is brown. Co, cornea; Le, lens; Tm, trabecular meshwork; st, corneal stroma; en, corneal endothelium; cp, lens capsule; ep, lens epithelium; fb, lens fiber cells; sc, Schlemm’s canal. (b and c) Confocal laser microscopic image (confocal) and Nomarski-differential interference microscopic image (diff) of human (b) and rat (c) eyes. Red shows the localization of NBC-1 and green shows nuclei. Cb, ciliary body; pg, pigmented ciliary epithelium.

Figure 3

The expression of NBC-1 isoforms in the eyes. (a) Peroxidase-antiperoxidase staining of the human cornea and trabecular meshwork. (b) Confocal laser microscopic image of the rat ciliary body, cornea, and lens. Details as in Figure 2.

Figure 4

The expression of NBC-1 mRNAs in HLE cells. RT-PCR analysis with (+) or without (–) the reverse transcription step (RT). Lanes 1 and 4: the common regions in NBC-1. Lane 2: the pNBC-1–specific region. Lane 3: the kNBC-1–specific region.

Figure 5

(a) pH_i recovery from acid in HLE cells. Results from two different experiments in the absence of HCO₃^–/CO₂. Cells were acidified by nigericin in solution a (Table 1); then the perfusate was changed to solution b. pH_i recovery was induced by solution a with or without 1 mmol/l amiloride (AML). (b) Three different experiments in the presence of HCO₃^–/CO₂. Cells were acidified in solution c; then the perfusate was changed to solution d. Thereafter, pH_i recovery was induced by solution c, solution c with 1 mmol/l AML, or solution c with 1 mmol/l AML and 0.3 mmol/l DIDS.

Figure 6

Effects of RZ-NBC on the expression of NBC-1 protein in HLE cells estimated by Western blot analysis. The Ab against NBC-1 C terminus was used, and a representative blot from three independent experiments is shown. Lane 1: nontransfected control cells. Lane 2: cells transfected with adenovirus vector carrying LacZ. Lane 3: cells transfected with adenovirus vector carrying RZ-MTP. Lane 4: cells transfected with adenovirus vector carrying RZ-NBC.

Figure 7

Effects of RZ-NBC on the transport activities in HLE cells. (a) pH_i recovery from acid load in the absence of Cl^–. Two different experiments from cells transfected with adenovirus vector carrying LacZ or adenovirus vector carrying RZ-NBC. (b) Na⁺-HCO₃^– cotransport activity determined as DIDS-sensitive pH_i recovery in the Cl^–-free HCO₃^– solution (solution e) containing 1 mmol/l amiloride. Cont, nontransfected control cells. Numbers of observations are 12 (Cont), 12 (LacZ), 10 (RZ-MTP), and 12 (RZ-NBC). ^*P < 0.001 compared with Cont. (c) Na⁺/H⁺ exchange activity determined as amiloride-sensitive pH_i recovery in solution e. Numbers of observations are 12 (Cont), 10 (LacZ), 9 (RZ-MTP), and 10 (RZ-NBC). dpH_i/dt, initial rate of pH_i recovery.

Figure 8

Possible roles of NBC-1 in the ocular tissues. Large arrows indicate the direction of net fluid transport. In the cornea (a) and lens (b), NBC-1 likely contributes to the net fluid transport. In the trabecular meshwork cells (c), NBC-1 may affect the contractility of these cells via interaction with L-type Ca²⁺ channels. In the ciliary epithelium (d), the contribution of NBC-1 to the net fluid transport remains to be established.