Temporal changes in the expression of mRNA of NADPH oxidase subunits in renal epithelial cells exposed to oxalate or calcium oxalate crystals

Abstract

Background: Exposure of renal epithelial cells to oxalate (Ox) or calcium oxalate (CaOx) crystals leads to the production of reactive oxygen species and cell injury. We have hypothesized that Ox and CaOx crystals activate NADPH oxidase through upregulation of its various subunits.

Methods: Human renal epithelial-derived cell line, HK-2, was exposed to 100 μmol Ox or 66.7 μg/cm(2) CaOx monohydrate crystals for 6, 12, 24 or 48 h. After exposure, the cells and media were processed to determine activation of NADPH oxidase, production of superoxide and 8-isoprostane (8IP), and release of lactate dehydrogenase (LDH). RT-PCR was performed to determine mRNA expression of NADPH subunits p22(phox), p40(phox), p47(phox), p67(phox) and gp91(phox) as well as Rac-GTPase.

Results: Exposure to Ox and CaOx crystals resulted in increase in LDH release, production of 8-IP, NADPH oxidase activity and production of superoxide. Exposure to CaOx crystals resulted in significantly higher NADPH oxidase activity, production of superoxide and LDH release than Ox exposure. Exposure to Ox and CaOx crystals altered the expression of various subunits of NADPH oxidase. More consistent were increases in the expression of membrane-bound p22(phox) and cytosolic p47(phox). Significant and strong correlations were seen between NADPH oxidase activity, the expression of p22(phox) and p47(phox), production of superoxide and release of LDH when cells were exposed to CaOx crystals. The expressions of neither p22(phox) nor p47(phox) were significantly correlated with increased NADPH oxidase activity after the Ox exposure.

Conclusions: As hypothesized, exposure to Ox or CaOx crystals leads to significant increases in the expression of p22(phox) and p47(phox), leading to activation of NADPH oxidase. Increased NADPH oxidase activity is associated with increased superoxide production and lipid peroxidation. Different pathways appear to be involved in the stimulation of renal epithelial cells by exposure to Ox and CaOx crystals.

Fig. 1

Relative levels of mRNA from real-time PCR of the membrane subunits of NADPH oxidase. Delta C value of membrane-bound subunit gp91^phox (A) and p22^phox (B).

Fig. 2

Relative levels of mRNA from real-time PCR of the cytosolic subunits of NADPH oxidase. Delta C values of cytosolic NADPH oxidase subunits p47^phox (A), p40^phox (B), p67^phox (C) and RAC-GTPase (D).

Fig. 3

NADPH oxidase production after HK monolayer cells exposed to oxalate (Ox) and CaOx monohydrate (COM) crystals.

Fig. 4

Effect of oxalate (Ox) and CaOx monohydrate (COM) crystals on HK-2 monolayer demonstrating extracellular production of SOD through conversion of WST-1 (2-(4-iodophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulpho-phenyl)-2 H-tetrazolium, monosodium salt) to a water-soluble formazan dye upon reduction with a superoxide anion.

Fig. 5

Effect of oxalate (Ox) and CaOx monohydrate (COM) crystals on HK-2 monolayer exhibiting cellular membrane injury through lipid peroxidation (8-IP).

Fig. 6

Effect of oxalate (Ox) and CaOx monohydrate crystals (COM) on HK-2 monolayer exhibiting cellular membrane injury through lactate dehydrogenase (LDH) release.