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Alterations in MDCK and LLC-PK1 cells exposed to oxalate and calcium oxalate monohydrate crystals
R L Hackett1, P N Shevock, S R Khan
1Department of Pathology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville 32610, USA.
Abstract:
Structural analysis of human kidney stones reveals the presence of cellular membranes and other cell fragments. Experimentally, calcium oxalate crystallization is facilitated when an exogenous nephrotoxin is given with ethylene glycol, thus providing cellular degradation products to act as heterogeneous nuclei. In this report, we tested whether oxalate alone could act as a cell toxin capable of producing damaged cells without the presence of an exogenous agent. Cultured LLC-PK1 and MDCK cells, when exposed to 1.0 mmol KOx, a concentration at the limit of metastability for calcium oxalate nucleation, were severely damaged as measured by specific lactate dehydrogenase (LDH) release in the spent media and by trypan blue exclusion. This effect was magnified by the addition of pre-formed calcium oxalate monohydrate crystals; the injury was significantly amplified when compared to exposure to oxalate alone. Scanning electron microscopy studies illustrated attachment of crystals to cells with loss of cell-to-cell and cell-to-substrate contact, as cells were released from the monolayer. In both oxalate and combined crystal-oxalate studies, more cells were released from the monolayer and exhibited considerably more damage when compared to controls. Oxalate, at the limit of metastability for calcium oxalate, is a cell toxin and can produce cellular degradation products. This effect is increased significantly by the addition of calcium oxalate monohydrate crystals.
Insights
Oxalate is a cell toxin that damages kidney cells, especially when calcium oxalate crystals are present. This finding suggests oxalate contributes to kidney stone formation by harming cells.
Area of Science:
- Nephrology
- Cell Biology
- Biochemistry
Background:
- Human kidney stones contain cellular material.
- Calcium oxalate crystal formation is accelerated by toxins and cell debris.
- Previous research used exogenous nephrotoxins to study stone formation.
Purpose of the Study:
- To investigate if oxalate alone can act as a cell toxin.
- To determine if oxalate can cause cellular damage and degradation products.
- To assess the synergistic effect of oxalate and calcium oxalate monohydrate crystals on cell viability.
Main Methods:
- Cultured LLC-PK1 and MDCK kidney cells were exposed to potassium oxalate (KOx).
- Cell damage was measured by lactate dehydrogenase (LDH) release and trypan blue exclusion.
- Scanning electron microscopy (SEM) was used to visualize cell-crystal interactions.
Main Results:
- Oxalate alone at 1.0 mmol KOx caused significant cell damage.
- The presence of calcium oxalate monohydrate crystals amplified oxalate-induced cell injury.
- SEM showed crystals attaching to cells, disrupting cell-cell and cell-substrate contacts, leading to cell detachment.
Conclusions:
- Oxalate acts as a cell toxin at concentrations near calcium oxalate nucleation metastability.
- Oxalate exposure leads to cellular degradation products.
- Calcium oxalate monohydrate crystals significantly enhance oxalate's cytotoxic effects on kidney cells.