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.

Localization of NBC1 Variants in Rat Kidney

“…The anti-kNBC1 antibody was raised against amino acids 4–16 of human kNBC1, and the anti-pNBC1 was raised against amino acids 2–12 of human pNBC1 [23]. We also produced the antibody against rb2NBC [9] according to the methods previously described [23].…”

“…We also produced the antibody against rb2NBC [9] according to the methods previously described [23]. In brief, a synthetic peptide corresponding to the C-terminal region of rb2NBC (amino acids 1079–1093) was coupled to C-terminal cysteine, conjugated to keyhole limpet hemocyanin, and the affinity-purified antibody was raised in rabbits.…”

“…In brief, a synthetic peptide corresponding to the C-terminal region of rb2NBC (amino acids 1079–1093) was coupled to C-terminal cysteine, conjugated to keyhole limpet hemocyanin, and the affinity-purified antibody was raised in rabbits. Western blot analysis was performed as described [23, 30]. In brief, the specimens were homogenized in an ice-cold buffer containing 280 m M sucrose and 0.2 m M Pefabloc SC (Boehringer Mannheim, Mannheim, Germany).…”

“…Mutations in NBC1 cause proximal renal tubular acidosis (pRTA) associated with ocular abnormalities, confirming the physiological importance of NBC1 [21,22,23,24]. Thus, we originally reported that R298S and R510H mutations in kNBC1 cause pRTA associated with band keratopathy, cataracts, and glaucoma [21].…”

“…In addition, a significant species difference exists in the expression patterns of NBC1 variants in pancreas [25]. To further clarify the relative roles of NBC1 variants in renal bicarbonate transport, we therefore performed the immunohistochemical analysis on rat kidney using the antibodies that can discriminate between kNBC1 and pNBC1 [23]. …”