Rat Renal Slices Research Articles

Concomitant lansoprazole ameliorates cisplatin-induced nephrotoxicity by inhibiting renal organic cation transporter 2 in rats.

Cisplatin is used widely for the treatment of multiple solid tumors. Cisplatin-induced nephrotoxicity is caused by renal accumulation of cisplatin via human organic cation transporter 2 (hOCT2). As lansoprazole, a proton pump inhibitor, is known to inhibit hOCT2 activity, lansoprazole might ameliorate cisplatin-induced nephrotoxicity. A previous study showed that concomitant lansoprazole administration ameliorated nephrotoxicity in patients receiving cisplatin. However, the detailed mechanism remains to be clarified. In the present study, the drug-drug interaction between lansoprazole and cisplatin was examined using hOCT2-expressing cultured cells and rat renal slices. Moreover, the effect of lansoprazole on cisplatin-induced nephrotoxicity and the pharmacokinetics of cisplatin in rats was investigated. In the uptake study, lansoprazole potently inhibited the uptake of cisplatin in hOCT2-expressing cultured cells and rat renal slices. The in vivo rat study showed that concomitant lansoprazole significantly ameliorated cisplatin-induced nephrotoxicity and reduced the renal accumulation of platinum up to approximately 60% of cisplatin alone at 72 h after cisplatin intraperitoneal administration. Furthermore, the renal uptake of platinum at 3 min after intravenous cisplatin administration in rats with cisplatin and lansoprazole decreased to 78% of rats with cisplatin alone. In addition, there was no significant difference in the plasma platinum concentration between rats treated with and without lansoprazole at 3 min after cisplatin intravenous administration. These findings suggested that concomitant lansoprazole ameliorated cisplatin-induced nephrotoxicity by inhibiting rOCT2-mediated cisplatin uptake in rats, thus decreasing cisplatin accumulation in the kidney. The present findings provided important information for the establishment of novel protective approaches to minimize cisplatin-induced nephrotoxicity.

Uptake of Cisplatin and its Metabolites Into Rat and Rabbit Renal Cortical Slices

The uptake of cisplatin and its metabolites into rat and rabbit renal cortical slices was investigated. Under anaerobic conditions the cisplatin uptake rate was significantly reduced by pretreatment with dinitrophenol and by organic cationic compounds (cimetidine, verapamil and quinidine). Verapamil competitively inhibited the uptake of cisplatin (apparent inhibitory constant (Ki) = 204.5 ± 43.0 μM). There was very little uptake of the cisplatin metabolite formed by complexation with glutathione. In addition, both aquated cisplatin and a metabolite formed by complexation with thiosulphate were taken up to a very low extent at 37°C compared with unchanged cisplatin. These results suggest that the intact chemical structure of cisplatin may be required for transport of cisplatin into the kidney and cisplatin may be partly transported via organic cation transport systems.

Inhibition of renin/prorenin receptor attenuated mesangial cell proliferation and reduced associated fibrotic factor release

The present study was designed to investigate the physiological roles of renin/prorenin receptor in cultured rat mesangial cells. The presence of renin/prorenin receptor in mesangial cells was determined by immunofluorescence staining, real-time quantitative reverse transcriptase-polymerase chain reaction (real-time PCR) and Western blotting assay. The expression of renin/prorenin receptors was identified in both rat renal slices and cultured mesangial cells. Meanwhile, the cultured mesangial cells were demonstrated synthesizing and secreting (Pro)renin as well. Knockdown of renin/prorenin receptor expression was performed by small interfering RNA (siRNA) transfection. Either knockdown or blockade of renin/prorenin receptor by handle region peptide (HRP) reduced mesangial cell proliferation. In the meantime, release of type IV collagen, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and mRNA level of transforming growth factor-β1 (TGF-β1) were decreased. On the other hand, both the production and activity of matrix metalloproteinase-2, a key enzyme to degrade type IV collagen, was increased. The present results indicated that renin/prorenin receptor played a regulatory role in mesangial cells proliferation and extracellular matrix accumulation. Blockade of renin/prorenin receptors may postpone over extracellular accumulation, which occurs in some pathological situations, via suppressed both mesangial cell proliferation and fibrotic factor release.

Activation of Protein Kinase Cζ Increases OAT1 (SLC22A6)- and OAT3 (SLC22A8)-mediated Transport

Organic anion transporters (OATs) play a pivotal role in the clearance of small organic anions by the kidney, yet little is known about how their activity is regulated. A yeast two-hybrid assay was used to identify putative OAT3-associated proteins in the kidney. Atypical protein kinase Czeta (PKCzeta) was shown to bind to OAT3. Binding was confirmed in immunoprecipitation assays. The OAT3/PKCzeta interaction was investigated in rodent renal cortical slices from fasted animals. Insulin, an upstream activator of PKCzeta, increased both OAT3-mediated uptake of estrone sulfate (ES) and PKCzeta activity. Both effects were abolished by a PKCzeta-specific pseudosubstrate inhibitor. Increased ES transport was not observed in renal slices from OAT3-null mice. Transport of the shared OAT1/OAT3 substrate, rho-aminohippurate, behaved similarly, except that stimulation was reduced, not abolished, in the OAT3-null mice. This suggested that OAT1 activity was also modified by PKCzeta, subsequently confirmed using an OAT1-specific substrate, adefovir. Inhibition of PKCzeta also blocked the increase in ES uptake seen in response to epidermal growth factor and to activation of protein kinase A. Thus, PKCzeta acted downstream of the epidermal growth factor to protein kinase A signaling pathway. Activation of transport was accompanied by an increase in V(max) and was blocked by microtubule disruption, indicating that activation may result from trafficking of OAT3 into the plasma membrane. These data demonstrate that PKCzeta activation up-regulates OAT1 and OAT3 function, and that protein-protein interactions play a central role controlling these two important renal drug transporters.

The Contribution of Organic Anion Transporters OAT1 and OAT3 to the Renal Uptake of Rosuvastatin

Rosuvastatin is a potent inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase and has been shown to be highly effective in reducing low-density lipoprotein cholesterol. Clinical trials have demonstrated that renal excretion and, in particular, tubular secretion, plays a role in rosuvastatin clearance. The aim of this study was to determine the involvement of the basolateral organic anion transporters, OAT1 and OAT3, in the renal uptake of rosuvastatin. Expression of human (h) OAT3 in Xenopus oocytes significantly increased the uptake of rosuvastatin above control levels (K(m) = 7.4 microM). In contrast hOAT1 did not mediate rosuvastatin uptake. Furthermore, hOAT3-mediated estrone-3-sulfate uptake could be inhibited, with a rank order of potency, by atorvastatin, rosuvastatin, simvastatin, and pravastatin, whereas hOAT1-mediated PAH uptake was only significantly inhibited by simvastatin. To estimate the contribution of hOAT3 to the overall renal uptake of rosuvastatin, a series of experiments were conducted using rat kidney slices. Rosuvastatin uptake in rat renal slices was abolished in the presence of the rat (r) Oat3-specific inhibitor benzylpenicillin, suggesting that rOat3 is responsible for the majority of rosuvastatin uptake across the basolateral membrane in rat kidney. From these findings, we can suggest that hOAT3 contributes to the renal uptake of rosuvastatin in humans.

Regulation of organic anion transporter 3 (OAT3) expression and function by membrane lipid raft‐associated proteins and cytoskeletons

In humans and rodents, two organic anion transporters (OATs), OATs 1 and 3, are expressed on the basolateral membrane of renal proximal tubules and mediate the secretion of exogenous or endogenous anions. Regulation of OAT1 and 3 has been reported in intact renal epithelia. For example, PKC activation inhibits organic anion uptake and EGF stimulates organic anion uptake mediated by OAT1 or OAT3. However, molecular mechanisms that regulate OAT expression and function are poorly understood. In the present study, plasma membrane lipid raft domains (LRDs) involved in protein trafficking and control of membrane processes were isolated from rat renal cortex and from HEK-293, human kidney epithelial cell line. Results indicate LRDs from rat renal cortical tissues contain caveolin 1, lipid-associated protein, and myosin – components previously implicated in protein sorting, trafficking, and cell signaling cascades. Similarly, LRDs from HEK-293 cells stably expressing human OAT3 (hOAT3) show co-localization of myosin and caveolin 1 with hOAT3. Cytoskeletal disruptors also alter hOAT3 expression and transport in HEK-293 cells, while a cholesterol depleting agent reduces OAT3 transport in rat renal slices. Thus, OAT3 expression and function may be regulated by LRDs and cytoskeletons. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.

Experimental study of renal disorder caused by oxaliplatin in rat renal cortical slices

Oxaliplatin is a newly developed antitumor platinum complex that is known to have low nephrotoxicity. The inhibitory effects of oxaliplatin on several tubular functions were compared with those of cisplatin and carboplatin, using a renal cortical slice system. Rat renal cortical slices were incubated with 0.25 mM to 2.0 mM of oxaliplatin, cisplatin, on carboplatin at 37 degrees C for 120 min. Para-amino hippuric acid (PAH) accumulation, gluconeogenesis, and ATP content in the rat renal slices were determined. PAH accumulation was not inhibited by carboplatin, but it was significantly inhibited by oxaliplatin and cisplatin. Inhibition of PAH accumulation by cisplatin was greater than that by oxaliplatin. Gluconeogenesis was not decreased by carboplatin, but it was suppressed by oxaliplatin and cisplatin in a dose-dependent manner. The decrease in gluconeogenesis induced by oxaliplatin was significantly greater than that induced by cisplatin. ATP content in the renal slices was decreased by oxaliplatin, cisplatin, and carboplatin to almost the same extent. As an in vivo experiment, 21.6 mmole/kg of oxaliplatin, cisplatin, or carboplatin was injected into rats; then blood urea nitrogen (BUN) and serum creatinine were determined on day 4. Significantly elevated levels of BUN and serum creatinine were observed only in the rats injected with cisplatin. Oxaliplatin did not cause nephrotoxicity in the in vivo study; however, the nephrotoxic pattern of oxaliplatin observed in the renal cortical-slice system resembled that of cisplatin. The reason why oxaliplatin is less nephrotoxic than cisplatin in vivo could not be fully elucidated in the present experiment using the renal cortical-slice system.

Characterization of renal excretion mechanism for a novel diuretic, M17055, in rats

M17055 was developed as a novel diuretic that inhibits both Na+, K+, and 2Cl− cotransport at the thick ascending Henle's loop and Na+ reuptake at the distal tubule. It is secreted at the renal proximal tubules. The purpose of the present study was to characterize the renal excretion mechanism of M17055. We used the renal cortical slices and brush border membrane vesicles (BBMVs) to investigate the transport mechanisms across the basolateral and brush border membranes, respectively. M17055 uptake by rat renal slices increased with time and was saturable. Several organic anions including probenecid, para-aminohippurate (PAH), and estrone-3-sulfate, decreased M17055 uptake. The uptake of M17055 was also observed into HEK293 cells expressing rat OAT1, and was inhibited by PAH. M17055 uptake by BBMVs was time-dependent, saturable, osmolarity-sensitive, and inhibited by several organic anions, but not by PAH. These results suggest that plural organic anion transport systems are involved in M17055 transport via both basolateral and brush border membranes of proximal tubule epithelial cells, a part of the renal uptake being mediated by OAT1.

Inhibition of gluconeogenesis and p -aminohipuric acid accumulation in rat renal cortical slices by ionic and nonionic contrast media

Renal dysfunction is a well-recognized complication induced by contrast media (CM). Nonionic CM have been introduced into clinical use to replace conventional ionic CM in an effort to reduce toxicity. However, the nephrotoxic effects of nonionic CM have not been fully evaluated. We previously determined the activities of N-acetyl-beta- d-glucosaminidase and gamma-glutamyltransferase released from rat and human renal slices incubated with contrast media. Dose-dependent enzyme release from renal slices was observed, but there was no statistical difference in the increase of enzyme activities between ionic and nonionic CM. The present experiment was conducted to compare the effects of ionic and nonionic CM on the metabolic function of rat renal slices. Rat renal cortical slices were incubated with ionic CM (diatrizoate, iothalamate) and nonionic CM (iopamidol, iohexol) at 37 degrees C for 90 min. To examine the dose-response effects of CM on gluconeogenesis and p-aminohipuric acid (PAH) accumulation in the rat renal slices, slices were incubated with 30, 60, and 90 mg I/ml of CM. The inhibitory effects of nonionic CM on gluconeogenesis and PAH accumulation were compared with those of ionic CM in an independent experiment, in which slices were incubated with CM at a concentration of 60 mg I/ml. In addition, rat renal slices were incubated with mannitol instead of CM to investigate the effects of osmotic pressure on gluconeogenesis and PAH accumulation. A dose-dependent reduction of gluconeogenesis in rat renal slices was demonstrated by both ionic CM and nonionic CM. The inhibition of PAH accumulation was dose-dependent with nonionic CM, but not with ionic CM. Gluconeogenesis and PAH accumulation within the renal slices were both inhibited according to the increase in osmotic pressure produced by mannitol. The reduction in gluconeogenesis and PAH accumulation within the rat renal slices incubated with 60 mg I/ml of nonionic CM were significantly less than those resulting from the same concentration of ionic CM. Nonionic CM is less nephrotoxic than ionic CM with regard to gluconeogenesis and PAH accumulation in rat renal slices. These differences in nephrotoxic effect between ionic and nonionic CM may in part be attributable to differences in osmotic pressure.

Nonionic contrast media are less nephrotoxic than ionic contrast media to rat renal cortical slices

Background: Nephrotoxicity induced by contrast media (CM) is well recognized. Nonionic CM with lower osmolality than that of conventional ionic CM have been developed in an effort to reduce toxicity. However, the nephrotoxic effects of nonionic CM have not been well evaluated. Although our previous experiments using rat renal cortical slices indicated that the direct cellular toxicity of nonionic CM is less than that of ionic CM, it was suggested that the less toxic effects of nonionic CM on the metabolic function of renal epithelial cells were in part attributable to the lower osmolality of nonionic CM. In the present experiment, the direct toxicity of nonionic CM on renal epithelial cells was compared with that of ionic CM under equiosmolar conditions, where the effects of osmotic pressure were excluded. Methods: Rat renal cortical slices were incubated with several kinds of CM at 37 °C for 120 min. Diatrizoate and iothalamate were employed as ionic CM. Iopamidol and iohexol were employed as nonionic CM. The activities of N-acetyl-β- d-glucosaminidase (NAG), γ-glutamyltransferase (GGTP), and lactate dehydrogenase (LDH) released from the renal slices into the incubation buffer were determined in order to evaluate renal epithelial damage caused by CM. Gluconeogenesis, p-aminohippuric (PAH) acid accumulation and ATP content in rat renal slices were determined with a view to examine the inhibitory effects of CM on the metabolic function of renal epithelial cells. The toxic effects of nonionic CM were compared with those of ionic CM under equiosmolar conditions, where mannitol was added to the experimental groups containing nonionic CM in order to exclude the effects of osmotic pressure. Results: A significant difference was generally not found with regard to enzyme release between ionic CM and nonionic CM plus mannitol. The inhibition of gluconeogenesis and PAH accumulation in rat renal slices by nonionic CM with mannitol was less than that by ionic CM. Although the ATP content was reduced by both ionic CM and nonionic CM plus mannitol, there was no significant difference between these two groups. Conclusions: The present experiments demonstrated that nonionic CM were less nephrotoxic than ionic CM with regard to the function of renal epithelial cells, including gluconeogenesis and PAH accumulation, under equiosmolar conditions. These differences in nephrotoxicity between ionic and nonionic CM cannot be fully attributable to differences in osmotic pressure.

Ex vivo stimulation of renal transport of the cytostatic drugs methotrexate, cisplatin, topotecan (Hycamtin) and raltitrexed (Tomudex) by dexamethasone, T3 and EGF in intact human and rat kidney tissue and in human renal cell carcinoma.

Previous experiments have shown that both in vivo and in vitro pre-treatment with various hormones increases the renal transport capacity for weak organic acids, such as PAH, in rats. The aim of the present study was to test whether or not accumulation of the anticancer drugs methotrexate (MTX), cisplatin (CP), raltitrexed (Tomudex) and topotecan (Hycamtin) can be increased in intact, healthy rat and human renal cortical slices and in human renal cell carcinoma (RCC). Intact, healthy human tissue was obtained from tumour bearing kidneys of patients suffering from RCC. Experiments were intended as a new approach to overcome so-called multidrug resistance. Kidney tissue slices were incubated for 24 h in William's medium E containing various concentrations of dexamethasone, T(3), or EGF. Thereafter slices were placed in anticancer drug containing Cross-Taggart medium and the drug uptake into kidney tissue was measured for 2 h. In intact rat and human renal tissue slices, the uptake of p-aminohippurate (PAH = reference substance) increased significantly after incubation in dexamethasone containing medium (134% and 156%, respectively). There were no stimulating effects of either T(3) or EGF on PAH accumulation. On the other hand, only the accumulation of MTX, but not of CP, raltitrexed or topotecan, was significantly enhanced after hormone pre-treatment both in intact renal tissue and in RCC. A stimulation of renal PAH accumulation can be performed ex vivo, as reported previously, both in intact rat and human renal cortical slices and in RCC. Discrepancies between the effects of dexamethasone and T(3) or EGF indicate different modes of action of these substances at the cellular level. Unfortunately, with the exception of MTX, the uptake of anticancer drugs can not be stimulated effectively ex vivo in human RCC tissue by the substances used. Evidently the transport of these anticancer drugs out of the kidney cells is more effective than their uptake.

Nephrotoxic effects of ionic and nonionic contrast media on rat and human renal cortical slices

Background. Nephrotoxicity is a well-known adverse effect of radiographic contrast medium (CM). Recently, several kinds of low- osmolality nonionic CM have been developed. However, the nephrotoxic effects of nonionic CM compared with those of ionic CM have not been well evaluated. Methods. To compare the direct nephrotoxic effects of ionic and nonionic CM on renal epithelial cells, rat and human renal cortical slices were incubated with CM at 37°C for 120 min. Diatrizoate and iothalamate were employed as ionic CM. Iopamidol, iohexol, iomeprol, ioversol, iopromide, and ioxilan were employed as nonionic CM. Direct toxicity of CM was evaluated by the activities of N-acetyl-β-D-glucosaminidase (NAG) and γ-glutamyl-transferase (GGT) released from the renal slices into the incubation buffer. Results. In the experiment with rat renal slices, NAG and GGT activities in buffer solutions were increased dose-dependently by 30, 60, and 90 mg I/ml of CM. There was no significant difference in NAG or GGT release between ionic and nonionic CM at the concentration of 60 mg I/ml. In the experiment with human renal slices, incubation with 60 mg I/ml of diatrizoate, iothalamate, iomeprol, and iopromide did not affect NAG levels. Significantly greater increases in NAG levels, compared with the control, were observed after incubation with iopamidol, iohexol, ioxilan and ioversol. Nevertheless, the increases in NAG caused by some of the nonionic CM were very slight. GGT release from human renal slices was significantly greater than that of the control in all experimental groups. Again, there was no significant difference in renal toxicity between ionic and nonionic CM. Conclusions. Newly developed nonionic CM had almost the same degree of nephrotoxicity against rat and human renal epithelial cells as conventional ionic CM, when direct renal toxicity was evaluated by enzyme release in an in-vitro experimental system using renal cortical slices.

P3B74 - Differences in atractyloside-induced lipid changes in precision-cut rat renal slices

P3B74 - Differences in atractyloside-induced lipid changes in precision-cut rat renal slices

In vitro stimulation of renal tubular p -aminohippurate transport by dexamethasone in rat kidneys and in intact kidney tissue of patients suffering from renal cell carcinoma

The aim of this study was to test whether or not the accumulation of p-aminohippurate (PAH) can be increased in intact human renal cortical slices obtained from tumor-bearing kidneys of patients suffering from renal cell carcinoma (RCC). Tissue slices were incubated for 24 h in Williams medium E containing 0.01-50 microM dexamethasone. Thereafter slices were placed in PAH-containing Cross-Taggart medium and PAH uptake into kidney tissue was measured for 2 h. In both rat and human renal tissue slices, PAH uptake capacity increased significantly in a concentration-dependent manner after 24 h of incubation in dexamethasone-containing medium (rat, 136%; man, 156%). The stimulatory effect was already significant after 12 h of incubation. In additional experiments it was shown that incubation in triiodothyronine (T3)-containing medium has different effects: in man, T3 does not influence the PAH accumulation capacity of renal cortical slices whereas in rats PAH accumulation is significantly lower after 24 h of incubation with T3. Thus stimulation of tubular transport capacity can be performed in vitro in human renal cortical slices. Discrepancies between the effects of dexamethasone and T3 indicate different modes of action of the two hormones at the cellular level.

Rat renal cortical slices: Maintenance of viability and use in in vitro nephrotoxicity testing

The majority of in vitro toxicology studies employing precision-cut rat renal slices are carried out using simple incubation systems over relatively short periods of time (usually up to 8 hr). We aimed to develop a system for longer-term (up to 24 hr) culture of rat renal slices. The viability of precision-cut rat renal cortical slices was therefore investigated under a variety of incubation conditions. Slices cultured using a dynamic organ culture system were found to be more viable than those cultured in shaken multiwell dishes. This improved viability was evident after only 2 hr of culture (as assessed by the measurement of LDH leakage). An oxygen enriched gaseous environment was found to be important for the maintenance of slice viability using the dynamic organ culture system and conditions were established which permitted the culture of viable slices for periods up to 24 hr. Using these optimized incubation conditions, less than 8% LDH leakage was observed at 24 hr and slices retained 84% of fresh ATP values. Histologically, few signs of slice degeneration were noted until 20 hr of culture. The renal slice system was shown to discriminate between the most toxic and least toxic of a series of cephalosporin antibiotics, based on measurements of slice ATP content.

Proteinic prorenin-releasing-stimulator (PRS) in the rat submandibular gland

The release of prorenin as well as renin from rat renal slices was confirmed by a rat prorenin-prosegment ELISA system and an assay system for determining the renin activity. A significant increase of the prorenin release was found by adding rat submandibular gland extract to the slice medium, indicating the existence of a prorenin-releasing stimulator (PRS) in the extract. The p I and molecular mass of PRS were 8.5–8.7 and 28–30 kDa, respectively. The PRS was completely inactivated by boiling or a proteinase treatment. Renin releasing stimulator, pro-; Cortical slice; Submandibular gland; ELISA, prorenin-prosegment; (Rat kidney)

An inwardly-directed sodium-amino acid cotransporter influences steady-state cell volume in slices of rat renal papilla incubated in hyperosmotic media

The effect of a neutral amino acid, 2-aminoisobutyric acid (AIB) on steady state cell volume has been examined in rat renal papillary slices incubated in hyperosmotic media (2,000 mosmol/kg H2O) containing high concentrations of NaCl and urea (thus imitating papillary interstitial fluid in the intact kidney during antidiuresis). Volumes were significantly increased (P less than 0.001) when external AIB was raised from 0.1 to 10 mmol/l. Na+-dependent AIB uptake occurred, and there were net increases in cell contents of Na+ and Cl-. Replacement of Na+ by Li+, but not by other cations, did not influence the effect of AIB concentration on cell volume, but this was abolished when Cl- was replaced by other anions. The effect of AIB was abolished by diphenylamine-2-carboxylate (10(-3) mmol/l), bumetanide (at 1 mmol/l but not 10(-2) mmol/l) and by N,N'-dicyclohexylcarbodiimide (0.5 mmol/l), but not by amiloride (1 mmol/l) or 4-acetamido-4'-iso-thiocyanato-stilbene-2,2'-disulphonic acid (1 mmol/l), and was enhanced by the presence of Ba2+ or quinine (1 mmol/l). The findings are interpreted in terms of an inwardly-directed Na+-amino acid cotransporter, which determines steady-state volume, requires simultaneous entry of Cl- through conductive pathways, and whose effects on cell volume are moderated by K+ efflux through volume-sensitive K+ channels.

Effect of Cisplatin on In Vitro Production of Lipid Peroxides in Rat Kidney Cortex

Cisplatin (c/s-diamminedichloroplatinum II), an antitumor agent with a dose-limiting adverse effect of nephrotoxicity, increased lipid peroxidation in a time- and concentration-dependent manner in rat renal slices incubated in vitro. The addition of an antioxidant, N-N'-diphenyl-p-phenylenediamine (DPPD), to the incubation medium completely inhibited this increase. We also studied the in vitro effects of agents that modify cisplatin nephrotoxicity on lipid peroxidation in the slices caused by cisplatin. Mannitol, which protects against cisplatin nephrotoxicity, almost completely inhibited the increase in lipid peroxidation caused by cisplatin. Methionine, which potentiates cisplatin nephrotoxicity, made the slices more susceptible to peroxidation. The decrease with cisplatin in p-aminohip-purate (PAH) accumulation in incubated kidney cortical slices, the accumulation being a representative biochemical process in the transport ability of renal cells, was partially inhibited when DPPD was in the medium. The results suggested that cisplatin directly affected renal tissues in which free radicals generated by cisplatin may interact with membrane lipids to cause the production of lipid peroxides that damage membrane function. Compounds that modify cisplatin nephrotoxity such as mannitol and methionine may act by affecting the production of renal lipid peroxides by cisplatin.

Excess mineralocorticoid receptor activity in patients with dexamethasone-suppressible hyperaldosteronism is under adrenocorticotropin control.

Total mineralocorticoid activity in human serum was assessed by the rat renal slice receptor assay (RRA). RRA values were compared to RIA-derived aldosterone (aldo) equivalents. Our data demonstrate that in normal subjects, mineralocorticoid receptor-binding steroids can be almost totally accounted for by immunoreactive deoxycorticosterone, corticosterone, cortisol, and aldo (RRA, 4.73 +/- 1.34 ng/ml aldo; RIA, 3.91 +/- 1.52 ng/ml aldo equivalents), while in 8 patients with dexamethasone-suppressible hyperaldosteronism (DSH), RRA values were greater than RIA values in the basal state (RRA, 7.57 +/- 0.75; RIA, 3.24 +/- 0.34; P less than 0.01). DSH patients had a RRA to RIA ratio after ACTH stimulation similar to the ratio in these patients in the basal state (basal, 2.34; ACTH-stimulated, 2.04). During dexamethasone treatment, RRA values fell markedly (1.82 +/- 0.26). Thus, total mineralocorticoid activity, as measured by RRA in DSH patients, was greater than RIA-measurable deoxycorticosterone, corticosterone, cortisol, and aldo, indicating the presence of an unidentified steroid which is dexamethasone suppressible and ACTH stimulable.

Renal Receptor-Binding Activity of Reduced Metabolites of Aldosterone: Evidence for a Mineralocorticoid Effect outside of the Classic Aldosterone Receptor System*

Reduced metabolites of aldosterone have been shown to have antinatriuretic and kaliuretic effects. We have studied the ability of four reduced metabolites of aldosterone to compete with [3H]aldosterone and [3H]dexamethasone for binding to the mineralocorticoid and glucocorticoid receptors of the kidney using adrenalectomized rat renal slices and cytosol, respectively, as sources of the binding proteins. 5 alpha-Dihydroaldosterone had 18.9% the ability to compete with [3H]aldosterone for binding to the cytoplasmic receptor of adrenalectomized rat renal slices in comparison to unlabeled aldosterone. Its antinatriuretic potency varied between 7-17%. Its ability to compete with [3H]dexamethasone for binding to the renal glucocorticoid receptor was only 1.9% in comparison to unlabeled dexamethasone. The relative competitive activities of 3 beta,5 alpha-tetrahydroaldosterone and 3 beta,5 beta-tetrahydroaldosterone with [3H]aldosterone to adrenalectomized rat renal slices cytosol were 1.26% and 0.05%, respectively, in comparison to unlabeled aldosterone. Their reported mineralocorticoid activities using the adrenalectomized rat bioassay (antinatriuresis) were 0.1-0.4% and 0.15%, respectively, in comparison to aldosterone. The most important aldosterone metabolite 3 alpha,5 beta-tetrahydroaldosterone showed negligible competitive activity with [3H]aldosterone or [3H]dexamethasone for the renal corticoid type I or type II receptors, respectively. However, this compound has been reported and confirmed to have weak but clear-cut mineralocorticoid activity (approximately 1/100th that of aldosterone). The mineralocorticoid activity of 3 alpha,5 beta-tetrahydroaldosterone cannot be explained by a mechanism involving the classic renal mineralocorticoid receptor. The mechanism could involve an alternative receptor system, a nonreceptor-mediated renal mechanism, or the conversion to a metabolite that would interact with classic receptors.