Influence of renal compensatory hypertrophy on mitochondrial energetics and redox status

A reduction in functional renal mass is common in numerous renal diseases and aging. The remaining functional renal tissue undergoes compensatory growth primarily due to hypertrophy. This is associated with a series of physiological, morphological and biochemical changes similar to those observed after uninephrectomy. Previous work showed that compensatory renal cellular hypertrophy resulted in an increase in susceptibility to several drugs and environmental chemicals and appeared to be associated with oxidative stress. Compensatory renal cellular hypertrophy was also associated with increases in mitochondrial metabolic activity, uptake of glutathione (GSH) across renal plasma and mitochondrial inner membranes, and intracellular GSH concentrations. Based on these observations, we hypothesize that the morphological, physiological and biochemical changes in the hypertrophied kidney are associated with marked alterations in renal cellular energetics, redox status and renal function in vivo. In this study, we used a uninephrectomized (NPX) rat model to induce compensatory renal growth. Our results show alterations in renal physiological parameters consistent with modest renal injury, altered renal cellular energetics, upregulation of certain renal plasma membrane transporters, including some that have been observed to transport GSH, and evidence of increased oxidative stress in mitochondria from the remnant kidney of NPX rats. These studies provide additional insight into the molecular changes that occur in compensatory renal hypertrophy and should help in the development of novel therapeutic approaches for patients with reduced renal mass.     

Influence of compensatory renal growth on susceptibility of primary cultures of renal cells to chemically induced injury.

Primary cultures of rat renal proximal tubular (PT) and distal tubular (DT) cells from control and uninephrectomized (NPX) Sprague-Dawley rats were established to study whether the altered toxicological responses identified in freshly isolated cells are maintained in culture. Previous work showed that primary cultures of PT cells from hypertrophied rat kidneys maintained their differentiated properties, as evidenced by their high respiratory rate, active transport function, transport and metabolism of glutathione, and their hypertrophic phenotype. In the present study, primary cultures of PT cells from NPX rat kidneys, but to a much lesser extent DT cells, were more susceptible to cellular injury induced by either mercuric chloride, KCN, or tert-butyl hydroperoxide (tBH), than corresponding cells from normal rat kidneys. Direct comparisons of cytotoxicity and lipid peroxidation induced by tBH in freshly isolated renal cells showed that the primary cultures of cells from NPX rat kidneys retained their altered susceptibility relative to cells from control rats. These results show that primary cultures of PT cells from NPX rats are more sensitive to cellular injury induced by three mechanistically distinct toxicants, demonstrating their usefulness in the study of the molecular and biochemical basis for the altered phenotype of compensatory renal growth. This is the first report validating the use of a mammalian renal cell culture model to study the toxicological effects of compensatory renal cellular hypertrophy.     

Adaptive changes in renal mitochondrial redox status in diabetic nephropathy

Nephropathy is a serious and common complication of diabetes. In the streptozotocin (STZ)-treated rat model of diabetes, nephropathy does not typically develop until 30 to 45 days post-injection, although hyperglycemia occurs within 24 h. We tested the hypothesis that chronic hyperglycemia results in a modest degree of oxidative stress that is accompanied by compensatory changes in certain antioxidants and mitochondrial redox status. We propose that as kidneys progress to a state of diabetic nephropathy, further adaptations occur in mitochondrial redox status. Basic parameters of renal function in vivo and several parameters of mitochondrial function and glutathione (GSH) and redox status in isolated renal cortical mitochondria from STZ-treated and age-matched control rats were examined at 30 days and 90 days post-injection. While there was no effect of diabetes on blood urea nitrogen, measurement of other, more sensitive parameters, such as urinary albumin and protein, and histopathology showed significant and progressive worsening in diabetic rats. Thus, renal function is compromised even prior to the onset of frank nephropathy. Changes in mitochondrial respiration and enzyme activities indicated existence of a hypermetabolic state. Higher mitochondrial GSH content and rates of GSH transport into mitochondria in kidneys from diabetic rats were only partially due to changes in expression of mitochondrial GSH carriers and were mostly due to higher substrate supply. Although there are few clear indicators of oxidative stress, there are several redox changes that occur early and change further as nephropathy progresses, highlighting the complexity of the disease.     

Protective effect of NADP-isocitrate dehydrogenase on the paraquat-mediated oxidative inactivation of aconitase in heart mitochondria

Protective role of NADP-isocitrate dehydrogenase in the oxidative inactivation of mitochondrial enzymes was analyzed. Administration of paraquat to the rat inactivated liver mitochondrial enzymes: the aconitase activity decreased to one quarter, and citrate synthase and fumarase to half, whereas cytosolic enzymes were not affected. Activities of heart mitochondrial and cytosolic enzymes were not at all changed in the rat treated with paraquat, but paraquat directly inactivated aconitase in the heart mitochondria isolated from the non-treated rats. The paraquat-dependent inactivation of aconitase was prevented by activating NADP-isocitrate dehydrogenase in the presence of oxidized glutathione. NADP-isocitrate dehydrogenase could regenerate glutathione in isolated heart mitochondria, indicating that paraquat-mediated inactivation depends on the glutathione-regenerating activity by enhanced NADPH supply. Lower NADP-isocitrate dehydrogenase activity in liver mitochondria cannot regenerate reduced glutathione for scavenging reactive oxygen species, resulting in the paraquat-induced oxidative inactivation of mitochondrial enzymes. However, higher activity of NADP-isocitrate dehydrogenase participates in the regeneration of reduced glutathione causing stabilization of enzymes in heart mitochondria.     

Role of organic anion and amino acid carriers in transport of inorganic mercury in rat renal basolateral membrane vesicles: influence of compensatory renal growth.

Susceptibility to renal injury induced by inorganic mercury (Hg(2+)) increases significantly as a result of compensatory renal growth (following reductions of renal mass). We hypothesize that this phenomenon is related in part to increased basolateral uptake of Hg(2+) by proximal tubular cells. To determine the mechanistic roles of various transporters, we studied uptake of Hg(2+), in the form of biologically relevant Hg(2+)-thiol conjugates, using basolateral membrane (BLM) vesicles isolated from the kidney(s) of control and uninephrectomized (NPX) rats. Binding of Hg(2+) to membranes, accounted for 52-86% of total Hg(2+) associated with membrane vesicles exposed to HgCl(2), decreased with increasing concentrations of HgCl(2), and decreased slightly in the presence of sodium ions. Conjugation of Hg(2+) with thiols (glutathione, L-cysteine (Cys), N-acetyl-L-cysteine) reduced binding by more than 50%. Under all conditions, BLM vesicles from NPX rats exhibited a markedly lower proportion of binding. Of the Hg(2+)-thiol conjugates studied, transport of Hg-(Cys)(2) was fastest. Selective inhibition of BLM carriers implicated the involvement of organic anion transporter(s) (Oat1 and/or Oat3; Slc22a6 and Slc22a8), amino acid transporter system ASC (Slc7a10), the dibasic amino acid transporter (Slc3a1), and the sodium-dicarboxylate carrier (SDCT2 or NADC3; Slc13a3). Uptake of each mercuric conjugate, when factored by membrane protein content, was higher in BLM vesicles from uninephrectomized (NPX) rats, with specific increases in transport by the carriers noted above. These results support the hypothesis that compensatory renal growth is associated with increased uptake of Hg(2+) in proximal tubular cells and we have identified specific transporters involved in the process.     

Diabetes increases susceptibility of primary cultures of rat proximal tubular cells to chemically induced injury

Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-l-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.     

The effects of 2,3,5-(triglutathion-S-yl)hydroquinone on renal mitochondrial respiratory function in vivo and in vitro: possible role in cytotoxicity.

Administration of 2,3,5-(triglutathion-S-yl)hydroquinone [2,3,5-(triGSyl)HQ] to rats causes severe renal proximal tubular necrosis. Although the cellular target(s) for 2,3,5-(triGSyl)HQ is not known, substantial evidence implicates mitochondria as the primary cellular target for aliphatic S-conjugates. To determine whether mitochondria are targets for 2,3,5-(triGSyl)HQ, the in vivo and in vitro effects of this conjugate on rat renal mitochondria (RRM) were investigated. In vitro exposure of RRM to 2,3,5-(triGSyl)HQ inhibited site I-supported respiration to a much greater extent than site II-supported respiration. Inhibition of mitochondrial function, as manifested by decreases in the respiratory control ratios, were a consequence of significant elevations in state 4 respiration. Inhibition of constitutive gamma-GT activity with AT-125 had no effect on the ability of 2,3,5-(triGSyl)HQ to decrease mitochondrial function. The effects of 2,3,5-(triGSyl)HQ on mitochondrial function in vivo were subsequently assessed. Shortly (0.5-2.0 hr) following administration of 2,3,5-(triGSyl)HQ (20 mumol/kg, iv) to rats, a significant elevation of state 4 respiration was observed. Thereafter (4-16 hr) state 4 respiration returned to control values and state 3 respiration became significantly depressed. A total collapse in RRM function occurred by 24 hr. The effects of 2,3,5-(triGSyl)HQ on state 4 respiration preceded significant elevations in blood urea nitrogen, which occurred at 8 hr. However, pretreatment of animals with probenecid, an inhibitor of organic anion transport, caused a significant decrease in the 2,3,5-(triGSyl)HQ-mediated elevations in state 4 respiration at 1 hr, without preventing the subsequent development of renal necrosis. In contrast, AT-125, which protected animals from 2,3,5-(triGSyl)HQ-mediated nephrotoxicity, had no effect on the early (1 hr) elevations in state 4 respiration but did prevent the later (8 hr) decreases in state 3 respiration. The data suggest that the early elevation in state 4 respiration observed in vivo is unlikely to contribute to 2,3,5-(triGSyl)HQ-mediated nephrotoxicity. The relationship between the decrease in state 3 respiration seen at later time points and the subsequent development of toxicity require further study before a cause and effect relationship can be determined.     

Oxidative and mitochondrial toxic effects of cephalosporin antibiotics in the kidney. A comparative study of cephaloridine and cephaloglycin

Cephaloridine and cephaloglycin are the two most nephrotoxic cephalosporins released for human use. Cephaloridine has been shown to produce both oxidative and mitochondrial respiratory injury in renal cortex in patterns of dose (or concentration) and time that are consistent with pathogenicity. Cephaloglycin also produces respiratory toxicity, and recent studies have provided evidence that this injury results from an inactivation of mitochondrial anionic substrate transporters. The abilities of cephaloglycin to produce oxidative changes and cephaloridine to block mitochondrial substrate uptake have not been examined yet. We therefore compared these two cephalosporins with one another and with cephalexin, which is not nephrotoxic, in the production of the following: (1) several components of oxidative stress or damage [depletion of reduced glutathione (GSH) and production of oxidized glutathione (GSSG) in renal cortex, inhibition of glutathione reductase in vitro, and production of the lipid peroxidation products malondialdehyde (MDA) and conjugated dienes (CDs) in renal cortex]; and (2) renal cortical mitochondrial toxicity [to both respiration with, and the transport of, succinate]. Cephaloridine depleted GSH and elevated GSSG in renal cortex, inhibited glutathione reductase, and increased both MDA in whole cortex and CDs in cortical microsomes and mitochondria. While cephaloglycin depleted GSH at least as much as did cephaloridine, it produced one-fifth as much GSSG and had little or no effect on glutathione reductase activity or on cortical MDA or microsomal CDs; cephaloglycin caused a transient small increase of mitochondrial CDs. Cephalexin produced no oxidative changes except for a slight increase of mitochondrial CDs comparable to that produced by cephaloglycin. Both cephaloridine and cephaloglycin, but not cephalexin, decreased the unidirectional uptake of, and respiration with, succinate in cortical mitochondria. We conclude that cephaloridine and cephaloglycin are both toxic to mitochondrial substrate uptake and respiration, but differ significantly in their generation of products of oxidation.     

Renal toxicity of perchloroethylene and S-(1,2,2-trichlorovinyl)glutathione in rats and mice: sex- and species-dependent differences

Suspensions of renal cells from rats and renal mitochondria from rats and mice were used to assess the sex and species dependence of acute toxicity due to perchloroethylene (Perc) and its glutathione conjugate S-(1,2,2-trichlorovinyl)glutathione (TCVG). A marked sex dependence in the acute cytotoxicity of both Perc and TCVG was observed: Perc caused significant release of lactate dehydrogenase (LDH) in isolated kidney cells from male but not female rats, and TCVG caused much more LDH release from male than female rat kidney cells. Assessment of toxicity in suspensions of isolated mitochondria from kidneys of male and female rats revealed a generally similar pattern of sensitivity, with mitochondria from males exhibiting significantly more inhibition of State 3 respiration and decrease of respiratory control ratio than mitochondria from females. Respiratory function in mitochondria from male and female mice, however, was also significantly inhibited by Perc or TCVG but exhibited little sex dependence in the degree of inhibition. Comparison with results from similar studies using the congener trichloroethylene and its glutathione conjugate suggested that Perc and TCVG are more potent nephrotoxicants. Neither Perc nor TCVG produced any significant effects on cytotoxicity or mitochondrial function in isolated hepatocytes from rats or in isolated liver mitochondria from rats or mice, suggesting that the liver is not a major acute target for Perc or its glutathione conjugate. Thus, many of the species-, sex-, and tissue-dependent differences in toxicity of Perc and TCVG that are observed in vivo are also observed in these in vitro models.     

Effects of thiabendazole (TBZ) on mitochondrial function in renal cortex of ICR mice.

The effects of thiabendazole (TBZ) on mitochondrial function of the renal cortex were investigated in ICR mice. Mice were given 1000 or 2000 mg TBZ/kg body weight by gavage and mitochondria were isolated from the renal cortex for the measurement of respiratory rates. The state 3 and DNP-uncoupled respiratory rates of renal cortical mitochondria were dose-dependently depressed at 6 hours after dosing. The depression of these respiratory rates of renal cortical mitochondria was more marked at 16 hours after dosing. There was no depression in these respiratory rates of renal cortical mitochondria at 3 hours after dosing, although renal cortical concentrations of TBZ were higher than those at 6 or 16 hours after dosing. Histochemical examination revealed that NAD-linked isocitrate dehydrogenase, a marker enzyme of mitochondria, was inhibited in renal cortical tubules at 16 hours after dosing of 1000 or 2000 mg TBZ/kg body weight. Furthermore, renal cortical ATP level was significantly decreased at 16 hours after dosing of 1000 or 2000 mg TBZ/kg body weight. The results indicate that administration of TBZ caused mitochondrial dysfunction in renal cortical tubules of mice.     

Renal metallothionein metabolism after a reduction of renal mass. II. Effect of zinc pretreatment on the renal toxicity and intrarenal accumulation of inorganic mercury.

In the present study we examined the effects of zinc pretreatment (to induce the renal synthesis of metallothionein) on the renal accumulation and intrarenal distribution of inorganic mercury in uninephrectomized (NPX) and sham-operated (SO) rats 24 h after the animals were given a 0.75, 1.0 or 1.5 mumol/kg intravenous (i.v.) dose of inorganic mercury. We also examined the effects of zinc pretreatment on the nephropathy induced by the three doses of inorganic mercury. Zinc was administered at a dose of 306 mumol/kg (20 mg/kg) subcutaneously (s.c.) in the form of zinc sulfate once daily for 2 consecutive days prior to the administration of inorganic mercury. Following zinc pretreatment, the renal accumulation of injected inorganic mercury increased in both NPX and SO rats treated with the three doses of inorganic mercury, but the increase was significantly greater in the NPX rats. The enhanced accumulation of mercury was associated with an altered pattern in the intrarenal distribution of mercury, particularly in the NPX rats. The increased renal accumulation of mercury in both the NPX and SO rats was due primarily to its increase in the renal cortex. We have recently found that the synthesis of metallothionein in the renal cortex increases in NPX and SO rats given zinc. Therefore, it appears that there is a relationship between the content of preinduced cellular metallothionein in the cortex and the content of mercury that accumulates in the cortex. Zinc pretreatment also prevented the nephropathy induced by the three doses of inorganic mercury from occurring in both the NPX and SO rats. We propose that some of the protection may be related to the altered intrarenal accumulation and distribution of mercury that occurs after pretreatment with zinc. Hepatic accumulation of mercury also increased in both groups of rats, but the increase again was significantly greater in the NPX rats. Our findings show clearly that a significant reduction in renal mass alters the hepatic and renal accumulation of mercury when zinc pretreatment is used to induce the renal and hepatic synthesis of metallothionein. In addition, our findings show that zinc pretreatment protects both normal and remnant kidneys in rats from the nephrotoxic effects of inorganic mercury.     

Altered intrarenal accumulation of mercury in uninephrectomized rats treated with methylmercury chloride.

We tested the hypothesis that the intrarenal accumulation of mercury in rats treated with methylmercury is altered significantly as a result of unilateral nephrectomy and compensatory renal growth. Renal accumulation of mercury was evaluated by radioisotopic techniques in both uninephrectomized (NPX) and sham-operated (SO) rats 1, 2, and 7 days after the animals received a nonnephrotoxic intravenous dose of methylmercury chloride (5 mg/kg Hg). At all times studied after the injection of the dose of methylmercury, the renal accumulation of mercury (on a per gram kidney basis) was significantly greater in the NPX rats than that in the SO rats. The increased accumulation was due to a specific increase in the accumulation of mercury in the outer stripe of the outer medulla. Renal cortical accumulation of mercury was similar in both the NPX and SO rats. The percentage of the administered dose of mercury that was present in the total renal mass of the NPX and SO rats ranged between 5 and 15, depending on the day that the renal accumulation was studied. Approximately 40-50% of the total renal burden of mercury in both the NPX and SO rats was in the inorganic form. However, only less than 1% of the mercury in blood was in the inorganic form at the three times accumulation was studied. Very little mercury was excreted in the urine by either the NPX or SO rats. Only about 2 to 3% of the administered dose of mercury was excreted in the urine in 7 days. By contrast, the cumulative fecal excretion of mercury over 7 days was substantial in the NPX and SO rats, and significantly more mercury was excreted in the feces by the NPX rats (about 19% of the dose) than by that in the SO rats (about 16% of the dose). In conclusion, our findings indicate that unilateral nephrectomy and compensatory renal growth cause a significant increase in the accumulation of mercury in the renal outer stripe of the outer medulla in rats exposed to methylmercury. In addition, the findings indicate that the fecal excretion of mercury is also significantly increased.     

Effects of in vivo treatment of rats with trimethyltin chloride on respiratory properties of rat liver mitochondria

Liver mitochondria isolated from rats treated in vivo with trimethyltin chloride show stimulation of respiration using glutamate/malate as substrate, and a transient inhibition on rates of respiration using palmitoyl-L-carnitine as substrate. This phenomenon was observed with both ADP- and FCCP-stimulated respiration. In contrast, rates of respiration by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride, following prior treatment with clofibrate, were inhibited when glutamate/malate was respiratory substrates. With palmitoyl-L-carnitine no effect of trimethyltin chloride was observed. In vitro treatment of rat liver mitochondria, or of rat liver homogenates, led to the expected, powerful inhibition of respiration. The synthesis of ATP by liver mitochondria isolated from rats treated in vivo with trimethyltin chloride was not inhibited compared to mitochondria isolated from control rats. Similarly, ATP synthesis by mitochondria isolated from rats treated with clofibrate, before treatment with trimethyltin chloride, was not inhibited. We, therefore, conclude that the powerful inhibitory effects of trimethyltin found in vitro, is not expressed in vivo during the first 36 hr following administration. In vivo treatment of rats with trimethyltin chloride caused a marked increase in hepatic levels of taurine and glycine, while levels of glutathione and glutamine were diminished. This is consistent with an enhanced oxidative stress in the liver. Our findings lead to the conclusion that increased oxidative stress, rather than inhibition of the mitochondrial ATPase, is a likely major cause of the in vivo toxic effects due to trimethyltin chloride.     

Diazinon-induced oxidative stress and renal dysfunction in rats

Diazinon (O,O-diethyl-O-[2-isopropyl-6-methyl-4-pyrimidinyl] phosphoro thioate), an organo-phosphate insecticide, has been used worldwide in agriculture and domestic for several years, which has led to a variety of negative effects in non target species including humans. However, its nephrotoxic effects and mechanism of action has not been fully elucidated so far. Therefore, the present study was aimed at evaluating the nephrotoxic effects of diazinon and its mechanism of action with special reference to its possible ROS generating potential in rats. Treatment of rats with diazinon significantly enhances renal lipid peroxidation which is accompanied by a decrease in the activities of renal antioxidant enzymes (e.g. catalase, glutathione peroxidise, glutathione reductase, glucose-6-phosphate dehydrogenase, glutathione S-transferase) and depletion in the level of glutathione reduced. In contrast, the activities of renal γ-glutamyl transpeptidase and quinone reductase were increased. Parallel to these changes, diazinon treatment enhances renal damage as evidenced by sharp increase in blood urea nitrogen and serum creatinine. Additionally, the impairment of renal function corresponds histopathologically. In summary, our results indicate that diazinon treatment eventuates in decreased renal glutathione reduced, a fall in the activities of antioxidant enzymes including the enzymes involved in glutathione metabolism and excessive production of oxidants with concomitant renal damage, all of which are involved in the cascade of events leading to diazinon-mediated renal oxidative stress and toxicity. We concluded that in diazinon exposure, depletion of antioxidant enzymes is accompanied by induction of oxidative stress that might be beneficial in monitoring diazinon toxicity.     

Protective effect of Phyllanthus fraternus against bromobenzene induced mitochondrial dysfunction in rat liver mitochondria

The objective of the current study was to investigate the protective effect of an aqueous extract of Phyllanthus fraternus (AEPF) against bromobenzene induced mitochondrial dysfunction in rat liver mitochondria. Administration of bromobenzene (10 mmol/kg body wt.) significantly decreased the rate of respiration (with glutamate + malate or succinate as substrates), abolished respiratory control ratio (RCR) and P/O ratios completely. There was a significant increase in the levels of lipid peroxides and protein carbonyls and a significant decrease in the total sulphydryl groups. The activities of antioxidant enzymes like catalase, glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD) were decreased. The levels of antioxidants like reduced and oxidized glutathione were significantly decreased compared to control. Administration of rats with an AEPF (100 mg/kg body wt.) prior to bromobenzene administration showed several beneficial effects like: (i) complete protection on mitochondrial respiration, RCR and P/O ratios (ii) lipid peroxides and protein carbonyl levels were significantly lowered (iii) increased the levels of sulphydryl groups and the activity of antioxidant enzymes and (iv) significant increase in the levels of reduced and oxidized glutathione. Vitamin E was used as positive control and bromobenzene induced mitochondrial dysfunction was protected better with AEPF compared to vitamin E.     

Cellular energetics and glutathione status in NRK-52E cells: toxicological implications

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived from rat proximal tubules. To assess toxicological implications of these properties, susceptibility to apoptosis induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a well-known mitochondrial and renal cytotoxicant, was studied. Cells exhibited high activities of several glutathione (GSH)-dependent enzymes, including gamma-glutamylcysteine synthetase, GSH peroxidase, glutathione disulfide reductase, and GSH S-transferase, but very low activities of gamma-glutamyltransferase and alkaline phosphatase, consistent with a low content of brush-border microvilli. Uptake and total cellular accumulation of [14C]alpha-methylglucose was significantly higher when cells were exposed at the basolateral as compared to the brush-border membrane. Similarly, uptake of GSH was nearly 2-fold higher across the basolateral than the brush-border membrane. High activities of (Na(+)+K(+))-ATPase and malic dehydrogenase, but low activities of other mitochondrial enzymes, respiration, and transport of GSH and dicarboxylates into mitochondria were observed. Examination of mitochondrial density by confocal microscopy, using a fluorescent marker (MitoTracker Orange), indicated that NRK-52E cells contain a much lower content of mitochondria than rat renal proximal tubules in vivo. Incubation of cells with DCVC caused time- and concentration-dependent ATP depletion that was largely dependent on transport and bioactivation, as observed in the rat, on induction of apoptosis, and on morphological damage. Comparison with primary cultures of rat and human proximal tubular cells suggests that the NRK-52E cells are modestly less sensitive to DCVC. In most respects, however, NRK-52E cells exhibited functions similar to those of the rat renal proximal tubule in vivo.     

Cholestasis induced by chronic treatment with alpha-naphthyl-isothiocyanate (ANIT) affects rat renal mitochondrial bioenergetics

Chronic cholestasis is characteristic of many human liver diseases. Renal injury has been often associated with this type of disease. The aim of this study was to evaluate the effect of cholestasis on kidney mitochondrial bioenergetics following in vivo chronic administration of alpha-naphthyl-isothiocyanate (ANIT), a known cholestatic agent. Serum markers of renal injury, kidney morphology and endogenous adenine nucleotides were measured in ANIT-treated rats (80 mg/kg per week s.c. for 16 weeks). Changes in membrane potential and mitochondrial respiration as well as alterations in mitochondrial calcium homeostasis were monitored. Cholestatic animals shown no alterations in renal morphology when compared with control. Additionally, following chronic ANIT administration, no significant alterations in mitochondrial respiratory function have been shown. The phosphorylation capacity of cholestatic kidney mitochondria was enhanced. Associated with these parameters, mitochondria from treated animals exhibited a decreased susceptibility to disruption of mitochondrial calcium homeostasis, due to permeability transition induction. These data suggest that, despite being submitted to chronic treatment with ANIT, kidney mitochondria from cholestasis-induced rats present some defense mechanisms to circumvent this aggression. They show improved phosphorylative capacity and, moreover, a decreased susceptibility to mitochondrial permeability transition induction, probably due to adaptative mechanisms of calcium transport.     

Renal cortical mitochondrial dysfunction upon cadmium metallothionein administration to Sprague-Dawley rats

A bolus dose of cadmium metallothionein (CdMT) produces renal proximal tubular dysfunction because it accumulates in the tubular epithelial cells and undergoes rapid degradation, releasing Cd. Morphologically, mitochondria appear to be the target organelle. The present study examined changes in renal cortical mitochondrial function following CdMT administration and investigated whether some of these effects could be ascribed to Cd2+ accumulation in the mitochondria. Sprague-Dawley rats were injected ip with 0.3 mg Cd as CdMT/kg and the animals were sacrificed after 6, 8, or 12 h. Two- to threefold increases in urinary protein excretion and LDH activity were evident at 8 h, with marked elevations (11- and 29-fold) thereafter. Renal cortical mitochondria were swollen and rounded at 12 h. The mitochondrial Cd level was 399 pmol/mg protein at 6 h and did not change significantly during the next 6 h; however, mitochondrial respiratory function declined with time. At 12 h, state 3 oxygen consumption, respiratory control ratio (RCR), and ADP:O (P/O) ratio were 48, 49, and 76% of control values, respectively, indicating inhibition of electron transfer and oxidative phosphorylation. The direct effect of Cd on mitochondrial function was examined by incubating mitochondria from untreated rats with 0.1-2 microM CdCl2. Rapid uptake of Cd resulted in concentration-dependent effects on respiration. After 1 min of incubation with 2 microM Cd, the mitochondria contained 262 microgCd/mg protein and state 3 respiration and RCR values were 75 and 33% of control levels, respectively. Thus, renal proximal tubular cell damage following a bolus dose of CdMT involves perturbations in mitochondrial respiration, brought on by the accumulation of Cd.     

Mitochondrial oxidative damage and myocardial fibrosis in rats chronically intoxicated with moderate doses of ethanol

Mitochondrial oxidative balance and myocardial fibrosis were investigated in pair-fed rats received ethanol (3%) or saccharose in drinking water for 8 weeks. The concentrations of glutathione, malondialdehyde, protein carbonyls and sulfhydrils were determined. The presence and distribution of fibronectin were detected by immunohistochemistry. The myocardial concentrations of reduced glutathione and protein sulfhydrils were lower in ethanol treated rats. The oxidised/reduced glutathione ratio, the levels of malondialdehyde and protein carbonyls were higher in ethanol-treated rats. The mitochondrial amount of proteins, glutathione and protein sulfhydrils were lower in ethanol treated rats, whereas the content of protein carbonyls and malondialdehyde were higher. Accumulation of fibronectin was detected at subepicardial and subendocardial districts in ethanol-treated rats, with moderate degree of fibrosis in 20% of the cases. In conclusion, moderate ethanol consumption is associated with oxidative damage to heart mitochondria and fibronectin deposition. These oxidative and ultrastuctural changes may be assumed as basic alterations in the development of alcoholic cardiomyopathy.