Accumulation of inorganic mercury along the renal proximal tubule of the rabbit

The purpose of the present study is to characterize the accumulation of inorganic mercury along the proximal tubule of the rabbit. New Zealand white rabbits were given a 0.5 mumol/kg dose of mercuric chloride along with 150 microCi of 203Hg. Forty-eight hours after the animals had been treated, individual segments of the nephron were obtained by microdissection. The segments of the nephron were measured in length and then were counted in a gamma counter to determine the percentage of the administered dose of inorganic mercury that had accumulated in them. There was significant accumulation of mercury along the proximal tubule during the 48 hr after the dose of mercuric chloride was administered. The S1 segment of the proximal tubule accumulated 0.000226 +/- 0.000031% (mean +/- SE) of the administered dose of inorganic mercury per millimeter tubule. The amount of mercury that accumulated in the S2 segment of the proximal tubule was similar to that in the S1 segment. By contrast, only half as much inorganic mercury accumulated in each millimeter of the S3 segment of the proximal tubule. No significant accumulation of inorganic mercury could be detected in pooled samples of various segments of the distal nephron. The findings in the present study indicate that the renal accumulation of inorganic mercury in the rabbit occurs mainly as a result of the accumulation of the metal in the proximal tubule, with the accumulation predominating in the S1 and S2 segments.     

Primary cultures of rabbit renal proximal tubule cells: II. Selected phase I and phase II metabolic capacities

Specific characteristics of cells vary as a function of time in culture. We have determined the stability of selected Phase I and Phase II biotransformation capacities in rabbit renal proximal tubule cells in primary culture. When grown in hormonally-defined medium, proximal tubule cells lost Phase I metabolic capacity. Cytochrome P-450 content and associated mixed-function oxidase activities present in kidney cortex microsomes were not detectable after 14 days in culture. Phase II glutathione-dependent metabolic functions were well retained in cultured cells compared with freshly isolated proximal tubules (FIPT). Cellular total glutathione content was 2.8 μg/mg protein in FIPT compared with approximately 10 μg/mg protein in stable confluent cultures. A higher total glutathione content of 20.6 μg/mg was noted in preconfluent cultures. The glutathione redox state was initially perturbed in FIPT with 37% of the total glutathione present found in its oxidized form. Tubule cells recovered to a normal ratio (6–13% of total glutathione in the oxidized form) while in culture. The glutathione S-transferase activity in 4-day-old cells in culture was reduced to 50% of the 4 U/mg protein level found in FIPT. No appreciable further decline in glutathione S-transferase activity was detected during 15 days in culture. The level of γ-glutamyl-transpeptidase (a brush-border enzyme necessary for glutathione uptake into proximal tubule cells) declined from 1499 mU/mg protein in homogenates of FIPT to 636 mU/mg in homogenates of 8-day-old cultured cells. A further decline in activity occurred during the next 7 days in culture. In conclusion, although Phase I metabolic functions were diminished in primary cultured rabbit proximal tubule cells, Phase II metabolic functions were retained at levels comparable with FIPT and well above those found in several established kidney cell lines.     

Statins inhibit aminoglycoside accumulation and cytotoxicity to renal proximal tubule cells

Nephrotoxicity due to renal proximal tubule accumulation of aminoglycoside (AG) antibiotics, such as gentamicin, represents a major clinical problem. Receptor-mediated endocytosis via the multi-ligand receptor megalin is thought to be a key mechanism in the cellular uptake of AGs and nephrotoxicity. This process can be modulated by the intracellular concentration of isoprenoid pyrophosphates derived from the processing of mevalonate by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Post-translation modifications by isoprenoid pyrophosphates are necessary for GTP-binding protein function. Given that statins inhibit HMG-CoA reductase and therefore affect the concentration of isoprenoid pyrophosphates, we have tested the hypothesis that statins will lead to a reduction in AG renal proximal tubule accumulation and cytotoxicity. Gentamicin accumulated within cultured proximal tubule derived opossum kidney (OK) cells and led to dose- and time-dependent cell death which was inhibited by non-toxic doses of simvastatin (IC₅₀ 1.3 μM), rosuvastatin (IC₅₀ 16.3 μM) and pravastatin (IC₅₀ 38.8 μM). The mechanism of inhibition was linked to the degree of cholesterol synthesis inhibition and GTP-binding protein unprenylation. Moreover, co-incubation with mevalonate or geranyl-geranyl pyrophosphate, products of HMG-CoA reductase, reversed the inhibitory effect of statins on cellular accumulation and cytotoxicity of gentamicin. In summary, our data suggest that the inhibition of the mevalonate pathway by statins may provide a potential therapeutic strategy to prevent AG-induced nephrotoxicity.     

Selective induction of apoptosis of renal proximal tubular cells caused by inorganic mercury in vivo

A recent notion, that a variety of toxicants causing necrosis can lead to apoptosis as well, has been demonstrated with cultured cells, but not with in an vivo system. In the present study, we examined the induction of both apoptosis and necrosis in the kidneys of Wistar rats exposed to mercuric chloride (HgCl(2)). A single injection of HgCl(2) to rats at a dose of 4 mg/kg resulted in an increase in the renal DNA fragmentation evaluated as an occurrence of apoptosis, prior to urinary excretion of alkaline phosphatase (ALP) and renal morphological changes assessed as necrotic phenomena. The mercury-promoted DNA fragmentation was induced in a dose-dependent manner. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and morphological observation of the nuclei revealed that apoptotic cells caused by HgCl(2) were predominantly found in the proximal tubules, but not in the distal tubules, glomeruli or medullary tubules. When we confirmed the proximal tubular-selective apoptosis by inorganic mercury with a combined technique of TUNEL staining with synchrotron radiation X-ray fluorescence (SR-XRF) imaging, it was shown that the apoptotic cells localized in the proximal tubules did contain higher level of mercury. Thus these results indicate that the proximal tubular cells-dominant site-specific distribution of mercury appears to be associated with induction of renal apoptosis and necrosis.     

Platinum complex-induced dysfunction of cultured renal proximal tubule cells

Platinum coordination complexes (PtCx) are potent against several types of cancer but are often nephrotoxic. With a view to developing a PtCx nephrotoxicity model, the toxicity of cisplatin (cDDP), transplatin (tDDP) and carboplatin (CBDCA) was studied in primary cultures of rabbit proximal tubule (RPT) cells and in the renal epithelial OK cell line. The cytotoxicity of these PtCx (10–3000 M) was assessed after 24 h exposure of confluent monolayers in terms of LDH release; their effects at non-cytotoxic concentrations (1–1000 M) on DNA and protein synthesis, glucose transport, marker enzymes and the total glutathione concentration were also determined, together with cellular platinum uptakes. The cytotoxicity ranking of the studied compounds differed for OK and RPT cells (cDDP>tDDP; cDDP>CBDCA and tDDP>cDDP; cDDP>CBDCA, respectively). Only results which were obtained in RPT cells corresponded to reported nephrotoxicity in vivo, making OK cells inappropriate for the study of PtCx nephrotoxicity in vitro. cDDP was about 10 times less cytotoxic for OK cells than for RPT cells because of lower cellular uptake. tDDP was unable markedly to inhibit biochemical and functional parameters in RPT cells below cytotoxic concentrations. At non-cytotoxic concentrations, cDDP and CBDCA depressed synthetic activity (mainly DNA) and, to a lesser extent, Na⁺-K⁺- ATPase activity and glucose transport in RPT cells. Total glutathione levels in RPT cells steadily increased during exposure to cDDP, tDDP and CBDCA, before the onset of cell death, arguing against an early role of glutathione depletion in PtCx toxicity. However, brush-border enzymes (-glutamyl transferase and alkaline phosphatase) and succinate dehydrogenase were insensitive to the action of either cDDP or CBDCA in RPT cells. On the basis of cytotoxicity and functional impairments, the toxicity of the three PtCx for RPT cells in vitro correlated with their reported nephrotoxicity in vivo, showing that RPT cells in primary culture are suitable for investigating the nephrotoxicity of PtCx.     

Intracellular effects of chronic arsenic administration on renal proximal tubule cells.

Arsenic is one of the more common toxic elements in the environment. The kidney accumulates this element and plays a major role in its metabolism and excretion. Mitochondria have been found in vitro to be highly sensitive to the toxicity of this element. Combined oxygen electrode and electron microscopic studies were conducted on kidneys of rats exposed to arsenate in the drinking water at concentrations of 40, 85, or 125 ppm for 6 weeks to evaluate in vivo mitochondrial toxicity. Decreased state 3 respiration and respiratory control ratios were observed in kidneys of rats given the 85 and 125 ppm dose levels. Ultrastructural alterations, which consisted of swollen mitochondria and increased numbers of dense autophagic lysosome-like bodies, were confined to proximal tubule cells of these same animals. This study places renal arsenate mitochondrial toxicity into an in vivo context and points to the value of using complementary techniques for assessing the subacute or chronic toxicity of environmental agents.     

Accumulation of BNP7787 in human renal proximal tubule cells

BNP7787, an investigational drug undergoing global Phase III development, appears to have potential advantages over other cytoprotective compounds that have been evaluated for preventing and mitigating cisplatin-induced nephrotoxicity. Herein, we characterized the in vitro accumulation of BNP7787 in human renal proximal tubule cells (HK-2) in which cisplatin is known to be taken up and accumulate. HK-2 cells were incubated with pharmacological concentrations of BNP7787 for varying times. Temperature-dependent accumulation of BNP7787 in cells was observed and the BNP7787-derived metabolite, mesna, formed intracellularly was directly monitored. The peak level of BNP7787-derived mesna measured in HK-2 cells was approximately 0.6 nmol/10(6) cells; this is pharmacologically similar to reported platinum concentrations in kidney cells and may be sufficient to afford nephroprotection. Therefore, in addition to previously suggested glomerular filtration, the cellular accumulation of BNP7787 by HK-2 cells is a plausible newly identified mechanism by which BNP7787 may accumulate in renal tubular cells, where it can exert its pharmacological effects to protect against cisplatin-induced nephrotoxicity by direct covalent conjugation of mesna with cisplatin, or by the formation of BNP7787-derived mesna-disulfide heteroconjugates that exert nephroprotective effects by inhibition of the key toxification enzyme targets γ-glutamyltranspeptidase and aminopeptidase N.     

Characterization of rabbit primary proximal tubule kidney cell cultures grown on Millicell-HA membrane filters

With the aim of developing a kidney cell culture system that can be used to assess renal toxicity in vivo, freshly isolated rabbit proximal tubules were plated on Millipore cellulose filters mounted in plastic inserts (Millicell-HA). DNA synthesis peaked on day 6 of culture and cells reached confluency by days 12–14. The integrity of the monolayer was confirmed by exclusion of [¹⁴C]inulin and cell viability demonstrated by linearity of protein synthesis over a 24-hr period. In confluent cultures, the organic anion, [¹⁴C]p-aminohippuric acid (PAH) and cation [¹⁴C]tetraethylammonium bromide (TEA) were shown to be transported from the basolateral to the apical side at a rate 5–6 times greater than that from the apical to basolateral side during the first 60 min of exposure. Probenecid decreased PAH transport by 60% and N-methylnicotinamide and quinine inhibited TEA transport by 40 and 56%, respectively. Uptake of [¹⁴C]-methylglucopyranoside into the cells was three times greater when label was added to the apical side than when label was added to the basolateral side. Apical uptake of glucose was sodium dependent and inhibited by more than 90% with phloridzin. Thus, kidney proximal tubule cells in the filter insert culture system display functional polarity which appears to mimic function in vivo and may be useful for examining mechanisms of nephrotoxicity.     

Multiplexed assay panel of cytotoxicity in HK-2 cells for detection of renal proximal tubule injury potential of compounds

Proximal tubules of the kidneys are one of the most common targets of nephrotoxic drugs and chemicals. Screens to predict nephrotoxic potential of compounds with insights to mechanisms of toxicity facilitate lead optimization, guide structure-activity relationships, minimize risks of clinical nephrotoxicity and therefore are valuable in the process of drug discovery. We developed and characterized an in vitro assay multiplexed to measure several endpoints of cytotoxicity using HK-2 cells. Assays for lactate dehydrogenase, cellular caspase 3/7 activation, resazurin dye reduction and Hoechst 33342 DNA staining were multiplexed to maximize the ability to detect cell injury. Assays were performed after 5- or 24-h incubations to further enhance the sensitivity of detection of toxicity. Individual assays were optimized for cell density, assay linearity and assay performance under multiplexed conditions. Inducers of apoptosis (staurosporine) and necrosis (perhexiline) were used to validate the mechanistic aspects of cell death. Nephrotoxic compounds (5-fluorouracil, gentamicin, cisplatin, acetaminophen, para-aminophenol, potassium dichromate, ibuprofen, doxorubicin, cyclosporine, citrinin, puromycin) were used to determine the potential of this method to detect proximal tubule toxicity of compounds. Overall, this cost-effective multiplexed platform is more sensitive than a single endpoint assay, provides mechanistic cues of toxicity and is amenable for higher throughput screening.     

Toxicity and transport of three synthesized mercury-thiol-complexes in isolated rabbit renal proximal tubule suspensions

Previous work has suggested that endogenous sulfhydryls, such as glutathione (GSH) and cysteine, are involved in the uptake and toxicity of HgCl2. To study this possibility, uptake and toxicity of synthesized Hg(SG)2, Hg(cysteinylglycine)2 [Hg(CYS-GLY)2] and Hg(CYS)2 were investigated in rabbit renal proximal tubule suspensions (RPT). The intracellular K+ was used as a toxicity indicator, and the mercury content in the tubules was measured by proton induced x-ray emission analysis. The toxicity rank order of the three synthesized mercury-thiol-complexes from the highest to the lowest was: Hg(CYS)2 > Hg(CYS-GLY)2 > Hg(SG)2. However, no significant difference among the mercury contents in the tubules exposed to these synthesized mercury-thiol-complexes was detected. Acivicin (0.25 mM), an inhibitor of gamma-glutamyltranspeptidase (GGT), decreased the toxicity of Hg(SG)2 in a manner that did not decrease the uptake of mercury in the tubules. This suggests that the toxicity of Hg(SG)2 requires processing to Hg(CYS-GLY)2 or Hg(CYS)2, while Hg(SG)2 may be taken up by the tubules via Na(+)-dependent GSH transporter since 10 mM acivicin, an inhibitor of this transporter dramatically decreased the uptake of Hg(SG)2. Organic anion transporter plays a minor role, if any, in the toxicity and uptake of Hg(SG)2 and Hg(CYS)2 since p-aminohippuric acid (PAH), an inhibitor of organic anion transporter, did not have significant effect on their uptake and toxicity. L-phenylalanine, an inhibitor of the neutral amino acid decreased the uptake of mercury, but to a lesser extent. This suggested that neutral amino acid transporter seemed to play a role, in part, in the toxicity and uptake of synthesized Hg(CYS)2. In summary, the data suggested that basolateral transport is important for the toxicity of the three synthesized mercury-thiol-complexes, and a variety of mechanisms are involved in the toxicity and uptake of these complexes in isolated rabbit RPT.     

Angiotensin II-mediated stimulation of phospholipase D in rabbit kidney proximal tubule cells

The present study was undertaken to demonstrate whether or not angiotensin II activates a phospholipase D in rabbit kidney proximal tubule cells. By measuring the formation of [3H]phosphatidic acid and [3H]phosphatidylethanol, we elucidate the direct stimulation of phospholipase D by angiotensin II. Angiotensin II leads to a rapid increase in [3H]phosphatidic acid and [3H]diacylglycerol, and [3H]phosphatidic acid formation preceded the formation of [3H]diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine by the action of phospholipase D, not from the action of diacylglycerol kinase on the diacylglycerol. In addition, the other mechanisms by which phospholipase D is activated was examined. We have found that phospholipase D was activated by extracellular calcium ion. It has also been shown that angiotensin II may activate phospholipase D through protein kinase C-independent pathway.     

Proliferative responses observed following vancomycin treatment in renal proximal tubule epithelial cells

Vancomycin (VAN) is a glycopeptide antibiotic used to treat gram-positive infections. Nephrotoxicity is a common side effect observed with vancomycin therapy. However, the mechanism of vancomycin-induced nephrotoxicity has not been fully characterized. In this study we examined the effect of vancomycin on cellular proliferation in renal proximal tubule cells. A dose- and time-dependent increase in cell number and total cellular protein was observed following vancomycin exposure. Vancomycin exposure also caused an increase in BrdU incorporation followed by the accumulation of renal proximal tubule cells in G₂/M phase of the cell cycle. These effects were inhibited by pretreatment with the mitogen-activated protein kinase inhibitor, PD098059, suggesting an association between the cell proliferative effect of VAN and the induction of the mitogen-activated protein kinase signaling pathway. Mitochondrial function in renal proximal tubule cells was assessed using oxygen consumption and ATP concentrations. We observed an increase in oxygen consumption and ATP concentrations following short-term exposure to vancomycin. Together, our data suggest that vancomycin treatment produces alterations in mitochondrial function that coincide with a cell proliferative response in renal proximal tubule epithelial cells.     

Mechanisms of absorption of inorganic mercury from rat small intestine. III. Comparative absorption studies of inorganic mercuric compounds in vitro

The transport of various inorganic mercuric compounds (HgX2s) was compared in everted intestinal sacs and intestinal brush border membrane vesicles (BBMV) of the rat. The preparations were incubated in a medium containing 10(-4) M HgX2 at pH 5.5, 6.4 or 7.4, respectively. The order of transport through the intestinal wall at each pH (HgOAc)2 greater than HgCl2 greater than Hg(SCN)2 greater than HgBr2 greater than Hg(CN)2) was the reverse order of their stability constants, and an increase in pH tended to increase the transport of HgX2. In the experiment with BBMV, similar results were obtained except for the transport of Hg(CN)2. These results suggest that the extent of transport of a certain HgX2 depends on its stability constant, and that the increase in pH promotes the transport possibly as a result of the conversion of HgX2 to Hg(OH)X and Hg(OH)2.     

Effect of organic and inorganic mercury on human sperm motility.

The effects of mercuric chloride and methyl mercuric chloride on the motility of human spermatozoa in vitro were investigated. Organic as well as inorganic mercury compounds decreased the percentage of motile spermatozoa. After 15 min. incubation with 40 microM mercuric chloride a significant decrease in sperm motility was observed. Less than 5% of spermatozoa were motile after 30 min. of exposure to 20 microM methyl mercuric chloride. These effects could not be attenuated by addition of 5 microM sodium selenite. The ultrastructural localization of mercury was demonstrated by autometallography. Silver-enhanced mercury deposits could be demonstrated only in spermatozoa exposed to inorganic mercury. In these cells mercury grains were most abundant in membranes of midpiece and tail.     

Effects of lead intoxication on intercellular junctions and biochemical alterations of the renal proximal tubule cells

Lead intoxication is a worldwide health problem which frequently affects the kidney. In this work, we studied the effects of chronic lead intoxication (500 ppm of Pb in drinking water during seven months) on the structure, function and biochemical properties of rat proximal tubule cells. Lead-exposed animals showed increased lead concentration in kidney, reduction of calcium and amino acids uptake, oxidative damage and glucosuria, proteinuria, hematuria and reduced urinary pH. These biochemical and physiological alterations were related to striking morphological modifications in the structure of tubule epithelial cells and in the morphology of their mitochondria, nuclei, lysosomes, basal and apical membranes. Interestingly, in addition to the nuclei, inclusion bodies were found in the cytoplasm and in mitochondria. The epithelial cell structure modifications included an early loss of the apical microvillae, followed by a decrement of the luminal space and the respective apposition and proximity of apical membranes, resulting in the formation of atypical intercellular contacts and adhesion structures. Similar but less marked alterations were observed in subacute lead intoxication as well. Our work contributes in the understanding of the physiopathology of lead intoxication on the structure of renal tubular epithelial cell–cell contacts in vivo.     

Comparison of organic and inorganic mercury distribution in suckling rat

Thiomersal is used as a preservative in vaccines given to small children. The metabolic product of thiomersal is ethylmercury and its distribution and kinetics are still not known, especially at this early age. The purpose of this study was to compare the body distribution of two forms of mercury: organic (thiomersal) and inorganic (mercury(2+) chloride) in very young, suckling rats. Mercury was applied subcutaneously three times during the suckling period on days 7, 9 and 11 of pups age, imitating the vaccination of infants. A single dose of mercury was equimolar in both exposed groups, i.e. 0.81 micromol Hg kg(-1). At 14 days of age the animals were killed and the total mercury analysed in blood and organs (kidney, liver and brain). The analytical method applied was total decomposition, amalgamation, atomic absorption spectrometry. The results showed that the level of mercury was higher in the liver and kidney of the inorganic mercury group than in the thiomersal exposed group. However, the brain and blood concentrations of mercury were higher in the thiomersal exposed group. These results need to be clarified by additional data on the kinetic pathways of ethylmercury compared with inorganic mercury.     

Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway

Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.     

Mechanism of cisplatin nephrotoxicity in rat renal proximal tubule suspensions

The effect of the nephrotoxic antineoplastic agent, cisplatin, on isolated rat renal proximal tubule suspensions was examined. [^195mPt]Cisplatin (31.7 μ ) uptake was progressive over a 5-hr incubation period to a maximum value of 8.39 nmol/mg protein. The addition of the metabolic inhibitors KCN and iodoacetic acid reduced cisplatin uptake at the later time points. The organic cation tetraethylammonium (1 m ) reduced [^195mPt]cisplatin (31.7 μ ) uptake to a greater extent than the organic anion p-aminohippuric acid (1 m ) suggesting preferential use of the cationic transport system by cisplatin. Biochemical perturbations observed included increased release of the lysosomal enzyme N-acetyl-β- -glucosaminidase as well as a time- and concentration-dependent increase in lipid peroxidation. Cisplatin inhibited both nystatin-stimulated and ouabain-sensitive respiration (Qo₂) after only 15 min of treatment. The biochemical changes observed were correlated with morphological alterations in the mitochondria. The results suggest that inhibition of oxidative phosphorylation may be a primary event in cisplatin-induced nephrotoxicity with later changes in lipid peroxidation and enzyme leakage.