Renal toxicity of ethylene glycol results from internalization of calcium oxalate crystals by proximal tubule cells

Abstract

Ethylene glycol exposure can lead to the development of renal failure due to the metabolic formation of calcium oxalate monohydrate (COM) crystals. The renal damage is closely linked to the degree of COM accumulation in the kidney and most likely results from a COM-induced injury to proximal tubule (PT) cells. The present studies have measured the binding and internalization of COM by primary cultures of normal PT cells from humans and from Wistar and Fischer-344 rats in order to examine the roles of these uptake processes in the resulting cytotoxicity. Internalization was determined by incubation of cells with [¹⁴C]-COM at 37 °C, removal of bound COM with an EDTA incubation, followed by solubilization of cells, as well as by transmission electron microscopy of COM-exposed cells. COM crystals were internalized by PT cells in time- and concentration-dependent manners. COM crystals were bound to and internalized by rat cells about five times more than by human cells. Binding and internalization values were similar between PT cells from Wistar and Fischer-344 rats, indicating that a differential uptake of COM does not explain the known strain difference in sensitivity to ethylene glycol renal toxicity. Internalization of COM correlated highly with the degree of cell death, which is greater in rat cells than in human cells. Thus, surface binding and internalization of COM by cells play critical roles in cytotoxicity and explain why rat cells are more sensitive to COM crystals. At the same level of COM accumulation after ethylene glycol exposure or hyperoxaluria in vivo, rats would be more susceptible than humans to COM-induced damage.

Introduction

Acute overdose ingestions of ethylene glycol (EG) can result in a renal failure that is linked with calcium oxalate monohydrate (COM) crystal accumulation in the kidney tissue (Corley et al., 2008, Cruzan et al., 2004, Jacobsen and McMartin, 1986). In these studies, COM accumulation is closely linked with necrotic damage—microscopically, necrotic damage is observed only in the presence of COM, and metabolically, kidney damage is correlated with the highest accumulation of COM. Renal accumulation of COM and renal parenchymal damage also occur in primary hyperoxaluria, i.e., from genetic defects in hepatic enzymes that normally metabolize precursors to other products, thereby shunting towards oxalate production as the major pathway (Milliner et al., 2001). A common animal model for formation of oxalate-containing kidney stones involves the administration of low doses of EG to rats to produce a chronic hyperoxaluria (Green et al., 2005, Asselman et al., 2003). Such doses can produce a large accumulation of COM in the kidney and accompanying renal damage (Corley et al., 2008, Cruzan et al., 2004), but do not produce the acidosis associated with acute EG overdose (Green et al., 2005). Interestingly, there is a large difference in sensitivity of certain rat strains to the renal toxicity of EG, where Wistar rats are much more susceptible than Fischer-344 (F344) rats (Cruzan et al., 2004, Li and McMartin, 2009).

In rats exposed to low levels of EG, the renal damage (a proximal tubule (PT) cell necrosis) is directly related to the amount of accumulation of COM in kidney tissue (Corley et al., 2008, Cruzan et al., 2004). The necrotic damage is most likely due to the accumulation of COM crystals, since, of the various EG metabolites, only oxalate or COM is cytotoxic to kidney cells in culture at relevant concentrations (Guo et al., 2007, Hackett et al., 1995, Scheid et al., 1995). More recent studies have convincingly demonstrated that COM crystals, and not the oxalate ion, are responsible for the cytotoxic effects of COM suspensions (Guo and McMartin, 2005, Schepers et al., 2005). Possible mechanisms for the COM-induced necrotic cell death include generation of oxidative stress (Khand et al., 2002, Scheid et al., 1996, Thamilselvan et al., 1997) as well as ATP depletion due to mitochondrial dysfunction (Cao et al., 2004, McMartin and Wallace, 2005). Although cell death could result from effects of COM at the cell membrane, uptake of COM by cells into the cytoplasm would allow for direct interactions with mitochondria or mitochondrial membranes. For example in isolated rat kidney mitochondria, COM, but not the oxalate ion, decreases mitochondrial respiration and directly induces the mitochondrial permeability transition (McMartin and Wallace, 2005), which would lead to a loss of the proton-motive force and to cell death.

In order for COM to accumulate in renal tissues, COM must adhere to tubular cell membranes, otherwise the newly formed crystals would be swept from the lumen by the natural flow (Finlayson and Reid, 1978). The binding of COM to epithelial layers provides an initiation spot for further binding and hence enlargement of crystals. Studies in transformed kidney cell lines have indicated that COM can bind to the plasma membrane of PT-like cells (Schepers et al., 2003, Verkoelen et al., 1999), while binding to distal tubular/collecting duct-like cells occurs only when the latter have been damaged (Verkoelen et al., 1998). Further studies have shown that COM crystals can be taken up into transformed kidney cells by an endocytotic process, with cytoplasmic appearance of crystals within 30–60 min (Lieske et al., 1994, Lieske and Toback, 1993). No studies have examined whether normal kidney cells are able to bind or internalize COM crystals, nor has the internalization of COM by cells been quantitated. Thus, the present studies have quantitatively examined the binding and internalization of COM by normal PT cells from humans and rats. Secondarily, these studies have compared the binding and internalization by cells from Wistar and F344 rats in order to examine whether these processes play roles in the strain difference in susceptibility to EG-induced damage. Through these studies, the key relationship between the internalization of COM and the amount of cell death produced by COM has been demonstrated.