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.

The effect of calcium on calcium oxalate monohydrate crystal-induced renal epithelial injury.

Since hypercalciuria is a common feature of idiopathic calcium oxalate (CaOx) nephrolithiasis, renal epithelial cells of stone patients are exposed to various crystals in the presence of high calcium. This study was performed to determine the effect of high calcium levels on CaOx crystal-induced cell injury. We exposed human renal epithelial cell line, HK2 in vitro to CaOx monohydrate crystals at a concentration of 133 microg/cm(2) for 1, 3, 6 or 12 h in the presence or absence of 5 or 10 mM/L calcium Ca(++). We determined the release of lactate dehydrogenase as marker of injury and hydrogen peroxide (H(2)O(2)) and 8-isoprostane (8-IP) as sign of oxidative stress. Cells were also examined after trypan blue and nuclear DNA staining with 4',6-diamidino-2-phenylindole to determine their membrane integrity and apoptosis respectively. Exposure of cells to 5 or 10 mM/L of Ca(++,) for up-to 6 h, resulted in increased trypan blue and DAPI staining and production of H(2)O(2). Similarly an exposure to CaOx crystals also resulted in increased trypan blue and DAPI staining and H(2)O(2) production. An exposure to 5 mM/L Ca or CaOx crystals also resulted in increased production of 8-IP. A combination of the two treatments, Ca and CaOx crystals, did not show anymore changes than exposure to high Ca or CaOx crystals alone, except in the case of a longer exposure of 12 h. Longer exposures of 12 h resulted in cells sloughing from the substrate. These results indicate that exposure to high levels of Ca or CaOx crystals is injurious to renal epithelial cells but the two do not appear to work synergistically. On the other hand, results of our earlier studies suggest that oxalate and CaOx crystals work in synergy, i.e., CaOx crystals are more injurious in the presence of high oxalate. Perhaps Ox and CaOx crystals activate different biochemical pathways while Ca and CaOx crystals affect the identical pathways.

Direct AFM measurements of adhesion forces between calcium oxalate monohydrate and kidney epithelial cells in the presence of Ca2+ and Mg2+ ions.

Adhesion forces between the calcium oxalate monohydrate (COM, whewellite) crystal and the layer of the epithelial kidney cells have been directly measured under buffer solutions by using atomic force microscope (AFM). Two renal epithelial lines, MDCK (a collecting duct line) and LLC-PK1 (a proximal tubular line), were used. All experiments were conducted in buffer solutions containing additional Ca(2+) and Mg(2+) ions in the various concentrations. For MDCK-cells, the obtained values of the adhesion force were in the range 0.12-0.51 nN and 0.12-0.20 nN for Ca(2+) and Mg(2+), respectively. No adhesion force (larger than 0.05 nN) has been found for LLC-PK1 cells. The "critical" concentrations of ions, near which the adhesion force (for MDCK-cells) was maximal, were found to be 100 mM. The "critical" concentration of ions and the tendency of the adhesion forces with the changing ions concentration, confirm earlier results of Lieske et al. [J.C. Lieske, G. Farell, S. Deganello, Urol. Res. 32 (2004) 117-123], in which the affinity (rather than the adhesion force) between the COM micro-crystals and the layer of the MDCK-cells were measured, calculating the radioactive signal of radioactive (14)C COM-crystals stuck to the cells. We believe that the aggregation of the COM crystals does not occur in the bulk urine due to short travel time through the nephron. If so, the kidney stone formation is determined by COM-seeding on the tubules walls. The further growth of the stone on the seed can take practically unlimited time because the COM crystal is practically is not soluble in water or urine solutions. The value of the adhesion force can be useful for evaluation of the adhesion energy or probability of the COM-aggregates to stick to the kidney epithelium under the urine flow. This probability is calculated taking into account the adhesion force, F(ad), and hydrodynamic driving force of the flow. This probability reflects the opportunity of the small aggregates to grow and form the kidney stones.

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 Randall's plaques to the papillary surface.

Dietary oxalate and calcium oxalate nephrolithiasis.

PURPOSE: Patients with calcium oxalate kidney stones are advised to decrease the consumption of foods that contain oxalate. We hypothesized that a cutback in dietary oxalate would lead to a decrease in the urinary excretion of oxalate and decreased stone recurrence. We tested the hypothesis in an animal model of calcium oxalate nephrolithiasis. MATERIALS AND METHODS: Hydroxy-L-proline (5%), a precursor of oxalate found in collagenous foods, was given with rat chow to male Sprague-Dawley rats. After 42 days rats in group 1 continued on hydroxy-L-proline, while those in group 2 were given chow without added hydroxy-L-proline for the next 21 days. Food and water consumption as well as weight were monitored regularly. Once weekly urine was collected and analyzed for creatinine, calcium, oxalate, lactate dehydrogenase, 8-isoprostane and H(2)O(2). Urinary pH and crystalluria were monitored. Rats were sacrificed at 28, 42 and 63 days, respectively. Renal tissue was examined for crystal deposition by light microscopy. RESULTS: Rats receiving hydroxy-L-proline showed hyperoxaluria, calcium oxalate crystalluria and nephrolithiasis, and by day 42 all contained renal calcium oxalate crystal deposits. Urinary excretion of lactate dehydrogenase, 8-isoprostane and H(2)O(2) increased significantly. After hydroxy-L-proline was discontinued in group 2 there was a significant decrease in urinary oxalate, 8-isoprostane and H(2)O(2). Half of the group 2 rats appeared to be crystal-free. CONCLUSIONS: Dietary sources of oxalate can induce hyperoxaluria and crystal deposition in the kidneys with associated degradation in renal biology. Eliminating oxalate from the diet decreases not only urinary oxalate, but also calcium oxalate crystal deposits in the kidneys and improves their function.

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

OBJECTIVE: To determine the differences in cell responses to synthetic and biological crystals of calcium oxalate (CaOx) and brushite MATERIALS AND METHODS: Nephrolithiasis depends on crystal retention within the kidneys, often promoted by crystal attachment to the injured renal epithelium; studies often use various crystals that might be injurious to cells and cause the exposure of crystal binding molecules on cell surfaces, thus promoting crystal attachment and retention. The synthetic crystals used in these studies might be more injurious than the biological crystals naturally produced in the kidneys and that form kidney stones. We exposed the renal epithelial cell line NRK 52E in vitro to CaOx or brushite crystals at 67 or 133 microg/cm(2) for 3 or 6 h. Synthetic crystals were purchased and the biocrystals were obtained by pulverizing CaOx and brushite stones. We determined the release of lactate dehydrogenase (LDH), hydrogen peroxide (H(2)O(2)) and 8-isoprostane (8-IP), and monocyte chemoattractant protein-1 (MCP-1), as markers of injury, oxidative stress and inflammation, respectively. Cells were also examined after trypan blue staining to determine their membrane integrity. We also examined crystals of CaOx by scanning electron microscopy both in the native state as well as after decalcification. RESULTS: Exposure to both the synthetic and biological crystals resulted in a significant increase in LDH release and trypan blue staining, as a sign of crystal-induced injury. There was increased production of H(2)O(2) and 8-IP, suggesting the development of oxidative stress. In addition MCP-1 production was also significantly increased. However, the synthetic crystals caused significantly higher increases in all the indicators than the biological crystals. CONCLUSIONS: These results indicate that even though both synthetic and naturally produced biocrystals invoke a response from the renal epithelial cells, the latter are significantly less injurious and inflammatory. Exposure to low concentrations of these crystals alone might not invoke an inflammatory response, cause the uncovering of crystal binding molecules on epithelial cell surfaces, and promote crystal attachment and retention.

Adhesion force between calcium oxalate monohydrate crystal and kidney epithelial cells and possible relevance for kidney stone formation.

AFM interaction force measurements have been performed between calcium oxalate monohydrate crystal (COM) colloidal probes and monolayers of renal epithelial cells (on a polymer substrate) in artificial urine (AU) solutions. The adhesion force was measured for the COM/MDCK cell interaction, while no adhesion force was found for the COM/LLC-PK(1) cell interaction. Long-range repulsive forces for both lines of cells were measured in the range of 2-3 mum. After removal of the cell from the substrate by the AU flow, the basal membrane (BM), with a thickness of 100-200 nm, remained on the substrate. In this case, the shorter-range repulsive forces were found on the extending (approaching) portion of force/indentation curves. Similar to the COM/MDCK cell interaction, the retracting portions of curves for COM/basal membranes have shown the existence of the attractive force of adhesion for the interaction of COM with a BM of MDCK cells, while no adhesion was found for COM/BM LLC-PK(1) cells interaction. No adhesion force was found for the interaction of a BM (of any cells) with the silicon nitride tip. Besides the hydrodynamic reasons, the adhesion difference between LLC-PK(1) and MDCK cells possibly explains the preferential deposition of crystals only in collecting ducts (lined with MDCK-type cells) and the lack of the crystal deposition in the proximal tubules (lined with LLC-PK(1)-type cells). Previous treatments of cells with oxalate alone increased the adhesion force COM/BM MDCK; however, even after oxalate treatment there was small or no adhesion between COM and BM LLC-PK(1) cells. Note that the adhesion force for COM/BM MDCK is practically independent of the probe velocity, i.e., does not have the viscous origin. Evaluation of the adhesion energy shows that this force should be related to the ionic or hydrogen bonds of samples.

Calcium phosphate-induced renal epithelial injury and stone formation: involvement of reactive oxygen species.

BACKGROUND: Crystal formation and retention are critical events for the formation of kidney stones. Oxalate and calcium oxalate (CaOx) crystals are injurious to renal epithelium, and membranes of injured cells promote crystal adherence and retention. Calcium phosphate (CaP) is the most common crystal in both urine and stones, most likely to form in the early segments of the nephron and can nucleate CaOx in a metastable solution. We hypothesized that CaP can also injure the renal epithelial cells. METHODS: We exposed proximal tubular origin line derived from pig proximal tubules (LLC-PK1), and collecting duct origin Madin-Darby canine kidney (MDCK) cell lines to various concentrations of Brushite (Br) crystals and investigated staining with Trypan Blue and the release of lactate dehydrogenase (LDH) into the medium as an indicator of injury. In order to determine the involvement of reactive oxygen species, we also measured LDH release in the presence of superoxide dismutase (SOD) and production of hydrogen peroxide (H2O2) and 8-isoprostane (8-IP) in the presence of the catalase. RESULTS: Exposure to Br crystals was associated with LDH release by both cell types, induced the production of H2O2 and 8-IP. Presence of SOD and catalase reduced LDH release as well as staining with trypan blue. Catalase was also associated with reduced production of H2O2 and 8-IP. CONCLUSION: Brushite crystals are injurious to cells of both the proximal tubules as well as collecting ducts. Injury is mediated by reactive oxygen species. We propose that CaP crystals can independently interact with renal epithelium, promote sites for crystal attachment, and then either grow into mature CaP stones or create sites for CaOx crystal nucleation, retention, and stone development.