William O. Berndt

Studies on the Mechanism of Nephrotoxicity and Nephrocarcinogenicity of Halogenated Alkenes

There is now a considerable weight of evidence from studies in a number of different laboratories with different haloalkenes to suggest that these compounds undergo conjugation with glutathione followed by degradation of the S-conjugate (Figure 1) to produce cytotoxic, and in some cases mutagenic, metabolites. These effects are dependent upon the sequential metabolism by gamma-glutamyl transferase and dipeptidases to produce the cysteine conjugates, and the presence of renal transport systems which concentrate the chemical in renal cells. These conjugates then appear to undergo further metabolism to a reactive thiol by the renal enzyme cysteine-conjugate beta-lyase, a process which can be blocked by inhibiting the enzyme with AOAA. Renal beta-lyase is present in both the cytosol and mitochondrial fractions, but toxicity studies in isolated cells and mitochondria indicate that the primary mode of action of these compounds is the inhibition of mitochondrial respiration, suggesting that the mitochondrial beta-lyase may be more important than the cytosolic enzyme in cysteine S-conjugate bioactivation. In addition to the renal cell injury caused by the presumed reactive thiol metabolite, reaction with DNA also occurs as the chlorinated, but not fluorinated, analogs are mutagenic, and in the case of HCBD, carcinogenic. Thus the target organ, cellular and subcellular specificity of haloalkene-S-conjugates, is due to the presence of bioactivating enzymes and the susceptibility of certain biochemical processes. The precise relationship between (1) the mitochondrial effects and cytotoxicity and (2) the interaction of the chemical with DNA and its mutagenicity require more precise understanding in order to elucidate the mechanism of S-conjugate-induced cell death and carcinogenicity. The routes and rates of metabolism of some of these compounds, with respect to glutathione conjugation vs. oxidative metabolism, in both experimental animals and man are required to help assess the risk associated with this class of chemicals.There is now a considerable weight of evidence from studies in a number of different laboratories with different haloalkenes to suggest that these compounds undergo conjugation with glutathione followed by degradation of the S-conjugate (Figure 1) to produce cytotoxic, and in some cases mutagenic, metabolites. These effects are dependent upon the sequential metabolism by gamma-glutamyl transferase and dipeptidases to produce the cysteine conjugates, and the presence of renal transport systems which concentrate the chemical in renal cells. These conjugates then appear to undergo further metabolism to a reactive thiol by the renal enzyme cysteine-conjugate beta-lyase, a process which can be blocked by inhibiting the enzyme with AOAA. Renal beta-lyase is present in both the cytosol and mitochondrial fractions, but toxicity studies in isolated cells and mitochondria indicate that the primary mode of action of these compounds is the inhibition of mitochondrial respiration, suggesting that the mitochondrial beta-lyase may be more important than the cytosolic enzyme in cysteine S-conjugate bioactivation. In addition to the renal cell injury caused by the presumed reactive thiol metabolite, reaction with DNA also occurs as the chlorinated, but not fluorinated, analogs are mutagenic, and in the case of HCBD, carcinogenic. Thus the target organ, cellular and subcellular specificity of haloalkene-S-conjugates, is due to the presence of bioactivating enzymes and the susceptibility of certain biochemical processes. The precise relationship between (1) the mitochondrial effects and cytotoxicity and (2) the interaction of the chemical with DNA and its mutagenicity require more precise understanding in order to elucidate the mechanism of S-conjugate-induced cell death and carcinogenicity. The routes and rates of metabolism of some of these compounds, with respect to glutathione conjugation vs. oxidative metabolism, in both experimental animals and man are required to help assess the risk associated with this class of chemicals.

The effect of depletion of nonprotein sulfhydryls by diethyl maleate plus buthionine sulfoximine on renal uptake of mercury in the rat

Rats pretreated with diethyl maleate (DEM, 3.37 mmol/kg, ip) and buthionine sulfoximine (BSO, 0.45 mmol/kg, ip) and subsequently given mercuric chloride (HgCl2, 0.014 mmol/kg, sc) had a significantly greater mortality rate over the 24 hr after injection than rats given only HgCl2 or HgCl2 following either DEM or BSO alone. Depletion of nonprotein sulfhydryls (NPSH) in the kidney significantly decreased mercury uptake in that organ. A similar effect was not seen in the liver despite marked depletion of NPSH. Similarly, there was a tendency for less in vitro mercury accumulation in renal cortical slices from rats made glutathione deficient by DEM + BSO compared to control, or rats made glutathione deficient by DEM or BSO alone. Depletion of nonprotein sulfhydryls by the combination of the depleting agents diethyl maleate plus buthionine sulfoximine (DEM + BSO) had a greater effect to alter organic ion accumulation in renal cortical slices than the agents alone. The higher mortality produced by mercuric chloride after DEM + BSO pretreatment may have been due to an increased availability of mercury in lethal concentrations at other organ sites. These data suggest the possible importance of NPSH in renal mercuric ion accumulation, but not in the liver.

The effect of unilateral nephrectomy on the nephrotoxicity of mercuric chloride in the rat.

Unilateral nephrectomy (UNX) induces a dramatic change in single-kidney structure and function. Therefore, the effects of nephrotoxins may be altered. To evaluate this possibility, mercuric chloride (2 mg/kg, sc) was given to male, Sprague-Dawley rats 2 days following either UNX or sham surgery. Nonoliguric acute renal failure developed and was qualitatively similar in both groups. Glomerular filtration rate (GFR) reached a nadir on Day 2 and was reduced to a greater extent in the UNX group. Furthermore, recovery of GFR was slower and occurred to a lesser extent by Day 10 in the animals subjected to UNX. Evidence of significant tubular dysfunction was present during the acute phase in both groups, as reflected by changes in the fractional excretion of sodium or lysozyme. Persistent tubular dysfunction was noted on Day 10 in both the sham and UNX groups, but the degree of dysfunction was greater in the UNX animals. The in vitro uptake of organic ions by renal cortical slices was reduced 24 hr following the injection of mercuric chloride although no difference was seen between the experimental groups. Mercury content within renal cortex was not increased in the UNX group at 1 or 3 hr but was higher 24 hr postinjection. Total urinary mercury excretion during the first day was not altered by UNX although single-kidney excretion was increased dramatically. These studies suggest that rats are more susceptible to mercuric chloride-induced nephrotoxicity 2 days following UNX. Although the mechanism(s) of this enhanced injury remains unclear, it does not appear to be completely related to an increase in renal cortical mercury content.

Potential involvement of renal transport mechanisms in nephrotoxicity.

Among the various physiological factors involved in the development of a nephrotoxic insult, certain renal transport systems may be important. The movement of exogenous organic anions and cations from the blood to the tubular fluid is well recognized. The anion transport system, which has been extensively characterized for the transport of p-aminohippuric acid, may have particular relevance. The nephrotoxic effect of citrinin, certain cephalosporin antibiotics and cysteine conjugates in rats and in isolated renal cells can be blocked by probenecid, a drug known to block the organic anion transporter competitively. The reduction in toxic response is correlated with the renal and cellular content of the nephrotoxic chemical.

Effects of fungal toxins on renal slice calcium balance.

Citrinin and ochratoxin A disrupt renal function in many animal species. The mechanism(s) underlying these actions is (are) unclear. Although citrinin has been shown to bind covalently to renal tissue, there also is evidence that it is active in the unmetabolized form. Altered calcium homeostasis has been suggested as an event which might mediate cell injury and/or death; a possible role for calcium in citrinin- or ochratoxin A-induced nephrotoxicity is reported here. Renal cortical slice calcium balance was monitored by the uptake of 45Ca. Either ochratoxin A or citrinin added to fresh renal cortex slices enhanced 45Ca accumulation. These effects were evident as early as 5 min after addition of the toxins. Greater 45Ca uptake occurred with bathing solution calcium concentration of 1.1 mM than in the absence of added carrier calcium. Finally, the effect of citrinin to reduce p-aminohippurate accumulation by renal cortical slices was greater in the presence of calcium than in its absence.

Renal and hepatic glutathione concentrations in rats after treatment with hexachloro-1,3-butadiene and citrinin

Renal and hepatic glutathione (GSH) concentrations were examined after treatment of male Sprague-Dawley rats with hexachloro-1,3-butadiene (HCBD) or citrinin alone and in combination, and after pretreatment with the GSH depleting agent diethylmaleate (DEM).It was found that both renal and hepatic GSH depletion were greater when either citrinin or HCBD was given following DEM. The effect was particularly striking when the doses used were so low as to be ineffective when given alone. When HCBD and citrinin were given in combination, the effect on GSH was approximately additive.Renal tubular organic ion transport in kidney slices was also compromised significantly when either citrinin or HCBD followed pretreatment with DEM. With HCBD, depression of tetraethylammonium (TEA) transport was seen after DEM; when given alone HCBD had no effect on TEA transport.

The effect of potassium dichromate and mercuric chloride on urinary excretion and organ and subcellular distribution of [203Hg]mercuric chloride in rats

The mechanism by which subthreshold/non-effective doses or concentrations of potassium dichromate (K2Cr2O7) potentiate the effect of moderately effective dose/concentrations of mercuric chloride (HgCl2) on renal organic ion transport is not understood. To investigate this effect, the rate of excretion of mercury from the intact animal and the renal and hepatic accumulation and subcellular distribution of mercury within kidney cortex following pretreatment or in vitro exposure to K2Cr2O7 were undertaken. Coincidental administration of K2Cr2O7 had no significant effect in altering the rate of excretion of labeled mercury. Organ distribution showed time dependence; however, the presence of K2Cr2O7 did not increase renal mercury concentrations. In fact, significantly less mercury was found at 4 h in the kidneys of rats receiving both metal salts. Subcellular distribution of labeled mercury (203Hg) was also not significantly altered by the presence of K2Cr2O7, although the distribution patterns for in vitro exposure and pretreated tissues were different. These studies show that K2Cr2O7 does not produce alterations in urinary elimination, organ distribution or subcellular distribution of mercury.

The effects of mercuric chloride on transport by brush border and basolateral membrane vesicles isolated from rat kidney

Both brush border and basolateral membrane vesicles were prepared from rat kidney by Percoll gradient centrifugation. The addition of mercuric chloride (100 nM) to vesicles prepared from healthy, male, Sprague-Dawley rats reduced p-aminohippurate (PAH) transport by basolateral vesicles. No effect was observed on glucose transport by brush border vesicles even at mercuric chloride concentrations as high as 10 microM. However, when the metal salt was added in the presence of 5% bovine serum albumin, basolateral PAH transport was unaffected. Transport studies also were done with vesicles isolated from rats pretreated with mercuric chloride (4 mg/kg, sc). Transport of PAH was unaffected at all times studied. Glucose transport was unaffected at 1 and 3 hr, but at 16 hr was reduced significantly. By 48 hr, brush border glucose transport had recovered. These data demonstrate that mercuric chloride can alter renal membrane function, and that the effects depend on the membrane vesicle population used. With pretreatment studies, the time after treatment also influences whether or not an effect is seen.

Ochratoxin/Citrinin as Nephrotoxins

The mycotoxins are a diverse group of secondary fungal metabolites. The diversity of chemical structure suggests that toxic mold metabolites may have the potential to cause diseases either after ingestion or contact on the skin. The mycotoxicoses that result from exposure to these compounds may be expressed as dysfunction of nervous system, the liver, the kidneys or potentially many other organs. Clearly, fungal infestation is not a requirement for the production of mycotoxicoses. Although the ability of certain mycotoxins to alter renal function in man has been debated only relatively recently, human contact with fungal toxins is not a new experience. Bagger (1931) had suggested that the earliest encounter of human mycotoxicoses were the ergotism episodes of the Middle Ages. It is likely that earlier occurrences also happened, but undoubtedly the frequency of such occurrences has decreased considerably in modern times. With the development of modern storage techniques for food, fungal contamination, as well as contamination by other microorganisms, has been greatly reduced and often is not considered a serious problem. Indeed, although human mycotoxicoses have not been ignored in recent times, it is nonetheless true that a much greater effort has been expended to address the problem of fungal contamination of animal feeds.

The effect of combined treatment with potassium dichromate and maleic acid on renal function in the rat

Subthreshold doses of potassium dichromate (K2Cr2O7) have been shown to enhance or potentiate the nephrotoxic effects of mercuric chloride and citrinin. The mechanism of this effect is unknown. The purpose of this study was to examine the phenomenon further by investigating the interaction of K2Cr2O7 with maleic acid, a nephrotoxicant with an action though to be different from those above. Male rats were housed in stainless steel metabolism cages and received a single intraperitoneal injection of maleic acid (300 mg/kg). K2Cr2O7 (10 mg/kg s.c.) or the combination of maleic acid plus K2Cr2O7. While treatment with either agent alone produced only minimal alterations in renal function, the combination of agents produced marked changes in 24 h urine volume, glucose excretion and osmolality. Water consumption was unaffected by the combined treatment. A marked decline in the capacity of renal slices to accumulate both organic anions (p-aminohippurate) and cations (tetraethylammonium) was observed after treatment with both substances. Modifications in renal and hepatic non-protein sulfhydryl content also were observed. These results suggest that K2Cr2O7, as was observed with both mercury and citrinin, enhanced or potentiated the nephrotoxic effects of maleic acid, probably in the proximal tubule, the principal site of glucose reabsorption and organic ion secretion.