Karen J. Byer

Free radical scavengers, catalase and superoxide dismutase provide protection from oxalate-associated injury to LLC-PK1 and MDCK cells

PURPOSE Current studies have provided evidence that exposure of renal epithelial cells to oxalate and calcium oxalate crystals induces lipid peroxidation and injures the cells. Since oxidant/antioxidant balance is likely to play a critical role, we determined the effect of antioxidant scavengers on production of free radicals and injury to LLC-PK1 and MDCK cells from exposure to oxalate (Ox) or Ox + calcium oxalate monohydrate (COM) crystals. MATERIALS AND METHODS LLC-PK1 and MDCK cells were grown in monolayers and exposed to 1.0 mmol. Ox or 1.0 mmol. Ox + 500 microg. /ml. COM crystals for 120 or 240 minutes. We measured the release of lactate dehydrogenase (LDH) as a marker for cell injury and malondialdehyde (MDA) as a marker of lipid peroxidation. Superoxide and hydroxyl radicals were measured in the presence or absence of 400 U/ml. catalase, or superoxide dismutase (SOD). RESULTS Exposure of LLC-PK1 cells to Ox resulted in a significant increase in MDA and release of LDH, which was further elevated when COM crystals were added. MDCK cells responded similarly to both challenges, but showed significantly less impact when compared with LLC-PK1 cells. Both treatments were associated with significant increase in the generation of hydroxyl and superoxide radicals by both cell types. In both cell lines, the addition of catalase or SOD significantly reduced the increase of MDA and release of LDH. CONCLUSIONS Results of the present study indicate that both Ox and COM crystals are injurious to renal epithelial cells and the injury is associated with generation of free radicals. Cells of proximal tubular origin are more susceptible than those of distal tubules and collecting ducts. Free radical scavengers, catalase and SOD provide significant protection.

EXPRESSION OF BIKUNIN mRNA IN RENAL EPITHELIAL CELLS AFTER OXALATE EXPOSURE

PURPOSE To determine the changes in expression of bikunin in renal epithelial cells on exposure to oxalate and calcium oxalate crystals. MATERIALS AND METHODS This study used reverse transcription polymerase chain reaction (RT-PCR) to examine bikunin mRNA expression levels in MDCK cells exposed to oxalate or calcium oxalate crystals. Poly(A)+ RNA was isolated directly from renal epithelial cells, then converted to cDNA with random primers and reverse transcriptase. To quantify the expression level ofbikunin mRNA, we developed a competitive DNA template for competitive PCR analysis. The PCR products were resolved by electrophoresis on 1.3% agarose gel and visualized with ethidium bromide. In this system, we could quantify the exact number of bikunin mRNA transcripts. Bikunin mRNA from rat liver was expressed as a positive control. RESULTS Bikunin mRNAs and competitive templates from renal epithelial cells were expressed in all samples as 434 bp and 312 bp bands, respectively. Bikunin expression was significantly increased in oxalate exposed cells. Cells exposed to calcium oxalate monohydrate crystals or latex beads showed no significant change in expression of bikunin. Western blotting analysis also showed increased expression of bikunin and inter-alpha-inhibitor-related proteins in the culture medium of oxalate exposed cells. CONCLUSIONS These findings suggest that renal epithelial cells express bikunin gene and have the capability to produce bikunin when stimulated by certain agents such as oxalate. This increased expression and production of bikunin may represent a protective response of renal epithelial cells to nephrotoxic challenges of oxalate.

Apatite induced renal epithelial injury: insight into the pathogenesis of kidney stones.

PURPOSE Kidney stone formation is associated with the deposition of hydroxyapatite as subepithelial plaques or tubular deposits in the renal papillae. We investigated the effect of renal epithelial exposure to hydroxyapatite crystals in vitro to develop an insight into the pathogenesis of kidney stones. MATERIALS AND METHODS NRK52E cells (No. CRL-1571, ATCC) were exposed to 67 or 133 microg/cm(2) hydroxyapatite (No. 21223, Sigma-Aldrich) or calcium oxalate monohydrate crystals (No. 27609, BDH Industries, Poole, United Kingdom). In some studies cells were also exposed to crystals from the basal side. After 3 or 6 hours of exposure medium was analyzed for lactate dehydrogenase, 8-isoprostane and H(2)O(2). Medium collected after cell exposure on the apical side was also analyzed for the production of monocyte chemoattractant protein-1 and prostaglandin E2. Cells were stained with DAPI to determine apoptotic activity and examined by scanning electron microscopy to observe crystal-cell interaction. RESULTS Cell exposure to hydroxyapatite resulted in H(2)O(2) and 8-isoprostane production as well as in lactate dehydrogenase release. Apical exposure appeared more provocative and injurious than basal exposure. Exposure to hydroxyapatite for 6 hours resulted in increased apoptotic activity. Apical exposure also resulted in increased monocyte chemoattractant protein-1 and prostaglandin E2 production. CONCLUSIONS Cell exposure to hydroxyapatite crystals induced oxidative stress and lipid peroxidation. It caused up-regulation of the inflammation mediators that may be responsible for the kidney inflammation in patients with stones that is associated with tubular hydroxyapatite deposition. It may also have a role in the eruption of subepithelial Randalls plaques to the papillary surface.

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

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.

Naturally produced crystals obtained from kidney stones are less injurious to renal tubular epithelial cells than synthetic crystals

To determine the differences in cell responses to synthetic and biological crystals of calcium oxalate (CaOx) and brushite

Oxalate induced expression of monocyte chemoattractant protein-1 (MCP-1) in HK-2 cells involves reactive oxygen species

Oxalate is a toxic end product of metabolism largely because of its propensity to crystallize and form calcium oxalate, which is insoluble at physiologic pH and often deposits at very unfortunate sites, notably the kidneys. In the current study, we investigated the oxalate-induced injury and up-regulation of monocyte-chemoattractant protein-1 (MCP-1) in HK-2 cells, a proximal tubular epithelial cell line derived from normal human kidney. The cells were exposed to oxalate ions for different lengths of time. The culture media was tested for LDH release, a cell injury marker. mRNA was isolated from the cells and subjected to reverse transcriptase-polymerase chain reaction. The data showed that oxalate exposure resulted in cell injury in a time and concentration dependent manner. The MCP-1 mRNA increased following exposure to oxalate and was reduced upon treatment with free radical scavengers, catalase and superoxide dismutase. These data support the importance of reactive oxygen species in the induction of expression of MCP-1 in renal epithelial cells. To our knowledge, this is the first report of MCP-1 expression and its upregulation by oxalate exposure in HK-2 cells.

Exposure of Madin-Darby Canine Kidney (MDCK) Cells to Oxalate and Calcium Oxalate Crystals Activates Nicotinamide Adenine Dinucleotide Phosphate (NADPH)-Oxidase

OBJECTIVE To investigate nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity in Madin-Darby canine kidney (MDCK) cells and the production of reactive oxygen species on exposure to oxalate (Ox) or calcium oxalate (CaOx) crystals. METHODS Monolayers of confluent Madin-Darby canine kidney cells were exposed to 100, 300, 500 μmol, 1 mmol Ox or 33, 66, 132 μg/cm(2) CaOx crystals for 15 minutes, 30 minutes, 1 hour, 2 hours, or 3 hours. After specified periods of exposure to Ox and CaOx crystals, lactate dehydrogenase release, trypan blue exclusion, activation of NADPH oxidase, and superoxide production were determined using standard procedures. The production of Nox4, a membrane associated subunit of the NADPH oxidase enzyme, was determined by western blot analysis. RESULTS Exposure to Ox and CaOx crystals leads to time- and concentration-dependent activation of NADPH oxidase. Western blot analysis showed an increase in the production of Nox4. The production of superoxide also changed in a time- and concentration-dependent manner, with maximum increases after 30-minute exposure to the highest concentrations of Ox and CaOx crystals. Longer exposures did not change the results or resulted in decreased activities. Exposure to higher concentrations also caused increased lactate dehydrogenase release and trypan blue exclusion indicating cell damage. CONCLUSION Results indicate that cells of the distal tubular origin are equipped with NADPH oxidase that is activated by exposures to Ox and CaOx crystals. Higher concentrations of both lead to cell injury, most probably through the increased reactive oxygen species production by the exposed cells.