Rat Renal Cortical Cells Research Articles

Trichlorophenol Induced Nephrotoxicity in Isolated Rat Renal Cortical Cells

Trichlorophenols (TCPs) are wood preservatives and intermediates in the synthesis of numerous pesticides. They appear in the environment as manufacturing byproducts and during the chlorination of drinking and waste water. TCPs are also metabolites of chlorinated compounds, such as chlorobenzenes and chlorophenoxy acids. Aquatic toxicity induced by these compounds has been studied in some detail, but few studies have focused on mammalian toxicity. The purpose of this study was to examine the nephrotoxic potential of four TCPs (2,3,4‐; 2,3,6‐, 2,4,5‐; and 2,4,6‐TCP) in an in vitro model using isolated renal cortical cells (IRCC) from male Fischer 344 rats. IRCC (~4 million cells/ml; 3 ml) were incubated with shaking at 37°C under a 95% oxygen/5% carbon dioxide atmosphere with a TCP (0 – 1.0 mM) or vehicle (dimethyl sulfoxide) for 60 min. General cytotoxicity was determined by assessing trypan blue exclusion of IRCC, measuring changes in lactate dehydrogenase (LDH) release, and quantitating ATP levels. LDH release was increased only at 1.0 mM for 2,3,6‐ and 2,4,6‐TCP. 2,3,4‐TCP increased LDH release at 0.5 mM or greater concentrations. However, 2,4,5‐TCP increased LDH release at 0.1 mM or greater concentrations. ATP levels were decreased at 1.0 mM for all compounds, at 0.5 mM for 2,3,4‐TCP and at all 2,4,5‐TCP concentrations. Therefore, the general order of decreasing nephrotoxic potential was 2,4,5‐TCP > 2,3,4‐TCP >2,3,6‐ and 2,4,6‐TCP.Support or Funding InformationSupported in part by NIH grant P20GM103434.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vitro nephrotoxicity induced by 2,4,5- and 2,4,6-trichloronitrobenzenes.

Chloronitrobenzenes are key chemical intermediates used in the manufacture of dyes, agricultural agents and industrial compounds. Although some data exists on the toxicity of mono- and dichloronitrobenzenes, there is a paucity of data on the toxicity profile of trichloronitrobenzenes (TCNBs). One of the target organs for mono- and dichloronitrobenzenes is the kidney. The purpose of this study was to examine the in vitro nephrotoxic potential of two TCNBs, 2,4,5-trichloronitrobenzene (2,4,5-TCNB) and 2,4,6- trichloronitrobenzene (2,4,6-TCNB), using freshly isolated rat renal cortical cells (IRCC), and to study potential mechanisms of bioactivation and toxicity. Briefly, IRCC were obtained from male Fischer 344 rats using a collagenase perfusion technique and incubated (~4 million cells/ml, 3 ml) with a TCNB (0.5 or 1.0 mM) or vehicle (dimethyl sulfoxide) for up to 90 min. In some experiments, cells were pretreated with an antioxidant (ascorbate, alpha-tocopherol, glutathione, N-acetyl-L-cysteine) or metabolizing enzyme inhibitor prior to a TCNB or vehicle treatment. Cytotoxicity was determined by measuring lactate dehydrogenase release. 2,4,6-TCNB induced cytotoxicity as early as 60 min and at 0.5 mM, while 2,4,5-TCNB did not induce cytotoxicity until 90 min at 1.0 mM. Antioxidant pretreatments were effective in reducing the toxicity induced by both TCNBS but were more effective against 2,4,5-TCNB. Cyclooxygenase inhibition reduced 2,4,6-TCNB, but not 2,4,5-TCNB, cytotoxicity, while inhibition of cytochrome P450, flavin monooxygenase and peroxidase activity were not protective. These results suggest 2,4,6-TCNB is more nephrotoxic than 2,4,5-TCNB and that free radicals contribute to TCNB nephrotoxicity in vitro.Supported in part by NIH grant P20GM103434.

Effect of cytochrome P450 isozyme inhibitors on 3,5‐ dichloroaniline nephrotoxicity in vitro

Chlorinated anilines are used in making agricultural chemicals, dyes, industrial compounds, and pharmaceuticals, and can induce nephrotoxicity in vivo and in vitro. 3,5‐Dichloroaniline (3,5‐DCA, 1.0 mM) induced nephrotoxicity in isolated rat renal cortical cells (IRCC) following 90 min exposure. IRCC pretreated with non‐selective cytochrome P450 (CYP) inhibitors partially attenuated 3,5‐DCA toxicity, suggesting that CYPs may play a role in 3,5‐ DCA bioactivation. The purpose of the this study was to further explore the role of CYP mediated 3,5‐DCA bioactivation. IRCC were obtained from male Fischer 344 rats and incubated (4 ×106 cells/ml; 3mL) with shaking (90 min) with dimethyl sulfoxide or 3,5‐DCA (1.0mM). IRCC were pretreated with various CYP inhibitors [isoniazid (1.0 mM), ketoconazole (0.1 mM), omeprazole (0.01 mM), diethyldithiocarbamate (DEDTCA; 0.1 mM), oleandomycin triacetate (0.5 mM), or sulfaphenazole (0.1 mM)] and cytotoxity was determined by measuring lactate dehydrogenase release. Pretreatment with DEDTCA, omeprazole, or sulfaphenazole partially attenuated 3,5‐DCA induced nephrotoxicity, while ketoconazole, isoniazid, oleandomycin triacetate had no effect. These results suggest that 3,5‐DCA is bioactivated via multiple pathways, including the CYP2C family. (Supported by NIH Grant 8P20GM103434 to West Virginia IDeA Network for Biomedical Research Excellence)

Regulation of Renal Organic Anion Transporter 3 (SLC22A8) Expression and Function by the Integrity of Lipid Raft Domains and their Associated Cytoskeleton

Background/Aims: In humans and rodents, organic anion transporter 3 (Oat3) is highly expressed on the basolateral membrane of renal proximal tubules and mediates the secretion of exogenous and endogenous anions. Regulation of Oat3 expression and function has been observed in both expression system and intact renal epithelia. However, information on the local membrane environment of Oat3 and its role is limited. Lipid raft domains (LRD; cholesterol-rich domains of the plasma membrane) play important roles in membrane protein expression, function and targeting. In the present study, we have examined the role of LRD-rich membranes and their associated cytoskeletal proteins on Oat3 expression and function. Methods: LRD-rich membranes were isolated from rat renal cortical tissues and from HEK-293 cells stably expressing human OAT3 (hOAT3) by differential centrifugation with triton X-100 extraction. Western blots were subsequently analyzed to determine protein expression. In addition, the effect of disruption of LRD-rich membranes was examined on functional Oat3 mediated estrone sulfate (ES) transport in rat renal cortical slices. Cytoskeleton disruptors were investigated in both hOAT3 expressing HEK-293 cells and rat renal cortical slices. Results: Lipid-enriched membranes from rat renal cortical tissues and hOAT3-expressing HEK-293 cells showed co-expression of rOat3/hOAT3 and several lipid raft-associated proteins, specifically caveolin 1 (Cav1), β-actin and myosin. Moreover, immunohistochemistry in hOAT3-expressing HEK-293 cells demonstrated that these LRD-rich proteins co-localized with hOAT3. Potassium iodide (KI), an inhibitor of protein-cytoskeletal interaction, effectively detached cytoskeleton proteins and hOAT3 from plasma membrane, leading to redistribution of hOAT3 into non-LRD-rich compartments. In addition, inhibition of cytoskeleton integrity and membrane trafficking processes significantly reduced ES uptake mediated by both human and rat Oat3. Cholesterol depletion by methyl-β-cyclodextrin (MβCD) also led to a dose dependent reduction Oat3 expression and ES transport by rat renal cortical slices. Moreover, the up-regulation of rOat3-mediated transport seen following insulin stimulation was completely prevented by MβCD. Conclusion: We have demonstrated that renal Oat3 resides in LRD-rich membranes in proximity to cytoskeletal and signaling proteins. Disruption of LRD-rich membranes by cholesterol-binding agents or protein trafficking inhibitors altered Oat3 expression and regulation. These findings indicate that the integrity of LRD-rich membranes and their associated proteins are essential for Oat3 expression and function.

4‐Amino‐2‐chlorophenol nephrotoxicity in vitro: alteration of cytotoxicity by antioxidants

4‐Amino‐2‐chlorophenol (4A2CP, 1.0 mM) induces nephrotoxicity in isolated rat renal cortical cells (IRCC). The purpose of this study was to examine potential metabolic pathways (cytochrome P450 [CYP], flavin mono‐oxygenase [FMO], and oxidation‐reduction) for bioactivation of 4A2CP. IRCC were obtained from male Fischer 344 rats and incubated in Krebs‐Henseleit buffer. IRCC (4 million cells/ml; 3 ml) were incubated with dimethyl sulfoxide (DMSO) or 4A2CP (1.0 mM) for 60 min with shaking. In some experiments, IRCC were pretreated with an FMO inhibitor [N‐octylamine (0.2 mM) or methimazole (1.0 mM)], CYP inhibitor [isoniazid (1.0 mM), ketoconazole (1.0 mM), or metyrapone (1.0 mM)], or an antioxidant [ascorbate (1.0 mM), glutathione (1.0 mM), N‐acetyl‐L‐cysteine (NAC, 2.0 mM), DPPD (0.05 mM), deferoximine (0.1 mM), or alpha‐tocopherol (1.0 mM or 2.0 mM)]. Cytotoxicity was assessed by measuring the release of lactate dehydrogenase (LDH). FMO or CYP inhibitors didn't decrease 4A2CP cytotoxicity. Only ascorbate, glutathione and NAC provided protection from 4A2CP toxicity. These results suggest that the mechanism by which 4A2CP induces toxicity does not involve activation by FMO's, CYP2E1, 2B or 3A4. Antioxidant protection suggests that a toxic metabolite is created via an auto‐oxidation process and/or oxidative stress plays a role in 4A2CP nephrotoxicity. (Supported by NIH Grant 5P20RR016477 to WV‐INBRE)

3,4‐Dichloronitrobenzene and 1,2,3‐trichloro‐5‐nitrobenzene in vitro nephrotoxicity in isolated rat renal cortical cells

Chloronitrobenzene (CNB) compounds are important chemical intermediates in the manufacture of dyes, drugs and agricultural chemicals. Some CNBs induce nephrotoxicity in animal models and human kidney cells in vitro. In this study, isolated renal cortical cells (IRCC) from male Fischer 344 rats were used to examine the nephrotoxic potential of two CNBs and to examine if the nephrotoxicity resulted from the formation of metabolites. Nephrotoxic potential was assessed by incubating 3,4‐dichloronitrobenzene (3,4‐DCNB) or 1,2,3‐trichloro‐5‐nitrobenzene (1,2,3 ‐TC5NB) at concentrations of 0 – 1.0 mM with IRCC (4 x 106 cells/ml) for 30 or 60 minutes and measuring lactate dehydrogenase (LDH) release. In some experiments, cells were pretreated with a cytochrome P450 inhibitor [piperonyl butoxide or metyrapone (1.0 mM)] or an antioxidant [glutathione (1.0mM), á‐tocopherol (1.0 mM), or ascorbate (2.0 mM)] followed by 0.5 mM 3, 4‐DCNB and 1, 2, 3‐TC5NB. Nephrotoxicity was induced by both CNBs (0.25 mM or higher) as early as 30 min. All antioxidant pretreatments reduced toxicity, suggesting that free radicals participate in the mechanism of CNB‐induced nephrotoxicity, while piperonyl butoxide and metyrapone attenuation of toxicity indicates metabolites play a role in the nephrotoxic mechanism. Supported by NIH Grant 3P20RR016477 to the West Virginia IDeA Network for Biomedical Research Excellence.

Intracellular ANG II directly induces in vitro transcription of TGF-β1, MCP-1, and NHE-3 mRNAs in isolated rat renal cortical nuclei via activation of nuclear AT1a receptors

The present study tested the hypothesis that intracellular ANG II directly induces transcriptional effects by stimulating AT(1a) receptors in the nucleus of rat renal cortical cells. Intact nuclei were freshly isolated from the rat renal cortex, and transcriptional responses to ANG II were studied using in vitro RNA transcription assays and semiquantitative RT-PCR. High-power phase-contrast micrographs showed that isolated nuclei were encircled by an intact nuclear envelope and stained strongly by the DNA marker 4',6-diamidino-2-phenylindole, but not by the membrane or endosomal markers. Fluorescein isothiocyanate-labeled ANG II and [(125)I]Val(5)-ANG II binding confirmed the presence of ANG II receptors in the nuclei with a predominance of AT(1) receptors. RT-PCR showed that AT(1a) mRNA expression was threefold greater than AT(1b) receptor mRNAs in these nuclei. In freshly isolated nuclei, ANG II increased in vitro [alpha-(32)P]CTP incorporation in a concentration-dependent manner, and the effect was confirmed by autoradiography and RNA electrophoresis. ANG II markedly increased in vitro transcription of mRNAs for transforming growth factor-beta1 by 143% (P < 0.01), macrophage chemoattractant protein-1 by 89% (P < 0.01), and the sodium and hydrogen exchanger-3 by 110% (P < 0.01). These transcriptional effects of ANG II on the nuclei were completely blocked by the AT(1) receptor antagonist losartan (P < 0.01). By contrast, ANG II had no effects on transcription of angiotensinogen and glyceraldehyde-3-phosphate dehydrogenase mRNAs. Because these transcriptional effects of ANG II in isolated nuclei were induced by ANG II in the absence of cell surface receptor-mediated signaling and completely blocked by losartan, we concluded that ANG II may directly stimulate nuclear AT(1a) receptors to induce transcriptional responses that are associated with tubular epithelial sodium transport, cellular growth and hypertrophy, and proinflammatory cytokines.

Nephrotoxicity induced by the R- and S-enantiomers of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and their sulfate conjugates in male Fischer 344 rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity characterized as polyuric renal failure and mediated via metabolites arising from oxidation of the succinimide ring. Recent findings have suggested that the stereochemical nature of NDPS metabolites may be an important factor in NDPS metabolite-induced nephrotoxicity. The purpose of the present study was to determine the role of stereochemistry in the in vivo nephrotoxicity induced by R-(+)- and S-(−)- N-(3,5-dichlorophenyl)-2-hydroxysuccinimide ( R- and S-NDHS) and the in vitro nephrotoxicity induced by their enantiomeric sulfate conjugates, R-(−)- and S-(+)- N-(3,5-dichlorophenyl)-2-hydroxysuccinimide- O-sulfate ( R- and S-NSC). Male Fischer 344 rats (four rats/group) were administered intraperitoneally (i.p.) an enantiomer of NDHS (0.05, 0.1 or 0.2 mmol/kg) or vehicle, and renal function monitored for 48 h. R-NDHS (0.1 or 0.2 mmol/kg) had little effect on renal function. In contrast, S-NDHS (0.1 mmol/kg) induced marked nephrotoxicity. The nephrotoxic potential of R- and S-NSC (0.5, 0.75 or 1.0 mM) was determined using freshly isolated rat renal cortical cells (IRCC, 3–4 × 10 6 cells/ml). Cytotoxicity was determined by measuring the release of lactate dehydrogenase (LDH) at the end of a 1 h incubation period. The LDH release observed in these studies was similar between R- and S-NSC. These results indicate that stereochemistry is an important factor for NDPS metabolite nephrotoxicity and that the role of stereochemistry, at least for NSC, occurs at extra-renal sites.

Effect of acetaminophen on the membrane anchoring of Na+, K+ATPase of rat renal cortical cells

In previous works we reported that the administration of a toxic dose of acetaminophen (APAP) induces acute renal failure (ARF) and promotes changes on Na(+), K(+)ATPase distribution in renal proximal plasma membranes. In the present work, we analyzed if APAP could promote the dissociation of Na(+), K(+)ATPase from its membrane anchorage. The participation of calpain activation was also evaluated. We analyzed the Triton X-100 extractability of Na(+), K(+)ATPase in freshly isolated cortical cell suspensions incubated with different APAP concentrations (0.1, 1, 10 and 100 mM). Both alpha(1) and beta(1) subunits were studied by Western blot. APAP promoted the increment of both subunits abundance in the Triton-soluble fraction. Calpain activation was detected in the membrane fractions of cells incubated with APAP. Incubation with APAP 0.1, 1 and 10 mM did not promote an increment in LDH release compared with controls, while APAP 100 mM promoted an increased LDH release. Our results show that incubation of proximal cells with sublethal and lethal APAP concentrations promotes the detachment of Na(+), K(+)ATPase from its membrane anchoring. Inhibition of calpain activation by SJA 7029 protected against APAP-induced membrane damage but not against APAP-induced increase of the Triton X-100 extractability of Na(+), K(+)ATPase.

Role of cytochrome P450 and glutathione S-transferase α in the metabolism and cytotoxicity of trichloroethylene in rat kidney

The toxicity and metabolism of trichloroethylene (TRI) were studied in renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. TRI was slightly toxic to both PT and DT cells, and inhibition of cytochrome P450 (P450; substrate, reduced-flavoprotein:oxygen oxidoreductase [RH-hydroxylating or -epoxidizing]; EC 1.14.14.1) increased TRI toxicity only in DT cells. In untreated cells, glutathione (GSH) conjugation of TRI to form S-(1,2-dichlorovinyl)glutathione (DCVG) was detected only in PT cells. Inhibition of P450 transiently increased DCVG formation in PT cells and resulted in detection of DCVG formation in DT cells. Formation of DCVG in PT cells was described by a two-component model (apparent V max values of 0.65 and 0.47 nmol/min per mg protein and K m values of 2.91 and 0.46 mM). Cytosol isolated from rat renal cortical, PT, and DT cells expressed high levels of GSH S-transferase (GST; RX:glutathione R-transferase; EC 2.5.1.18) α (GSTα) but not GSTπ. Low levels of GSTμ were detected in cortical and DT cells. Purified rat GSTα2–2 exhibited markedly higher affinity for TRI than did GSTα1–1 or GSTα1–2, but each isoform exhibited similar V max values. Triethyltinbromide (TETB) (9 μM) inhibited DCVG formation by purified GSTα1–1 and GSTα2–2, but not GSTα1–2. Bromosulfophthalein (BSP) (4 μM) only inhibited DCVG formation by GSTα2–2. TETB and BSP inhibited approximately 90% of DCVG formation in PT cytosol but had no effect in DT cytosol. This suggests that GSTα1–1 is the primary isoform in rat renal PT cells responsible for GSH conjugation of TRI. These data, for the first time, describe the metabolism of TRI by individual GST isoforms and suggest that DCVG feedback inhibits TRI metabolism by GSTs.

Binding affinity of sarpogrelate to 5-HT 2A receptor ligand recognition sites in rat renal cortical and mesangial cells in culture

We detected specific binding of 3H-ketanserin (0.6 nM) in rat renal cortical membrane preparations (4.70 ± 0.57 fmol/mg protein) and mesangial cells (7.55 ± 0.92 fmole/10 6 cells). Thus, the value in the renal cortical membrane corresponded to 15% of that in the cerebral cortical membranes (30.0 ± 2.9 fmole/mg protein). The affinity of 3H-ketanserin binding displacement activities by sarpogrelate, a selective 5-HT 2A receptor antagonist, in the renal cortical membrane (IC 50; 0.448 ± 0.061 μM) and mesangial cells (IC 50; 0.656 ± 0.187 μM) were almost 100-fold less than that in the cerebral cortical membrane (IC 50; 4.62 ± 1.02 nM). In the renal cortical membranes and mesangial cells, methysergide displaced a tiny fraction of 3H-ketanserin binding at concentrations up to 10 μM. These results did not explain the functional activity of 5-HT in the mesangial cells, and we conclude that specific 3H-ketanserin binding sites in the mesangial cells consisted of methysergide-resistant and non-serotonergic sites with low affinity for sarpogrelate.

DNA–Protein Crosslinks Induced by Nickel Compounds in Isolated Rat Renal Cortical Cells and Its Antagonism by Specific Amino Acids and Magnesium Ion

Suspensions of isolated renal cortical cells in modified Krebs-Henseleit buffer (pH 7.4) were incubated with nickel chloride, nickel acetate, nickel sulfate, and nickel subsulfide (0–2 mM) at 37°C for 2 h. A significant increase (63%) in DNA–protein crosslinks was observed at 2 mM nickel sulfate, whereas nickel subsulfide induced a significant increase in such crosslinks beginning at 0.5 mM concentration and a maximum increase of 200% of the control value reached at 2 mM concentration. No significant reduction in viability of renal cortical cells (as measured by trypan blue exclusion) was observed due to these nickel compounds at any concentration used. In the second series of experiments, coincubation of nickel subsulfide (2 mM) withl-histidine (8 or 16 mM),l-cysteine (4 or 8 mM), orl-aspartic acid (8 or 24 mM) significantly reduced the DNA–protein crosslinks induced by 2 mM nickel subsulfide. Similarly Mg2+(24 mM), but not Ca2+(24 mM), was able to antagonize nickel subsulfide-induced increase in DNA–protein crosslinks. High extracellular levels of Mg2+and these amino acids significantly decreased the accumulation of Ni2+from nickel subsulfide in renal cortical cells. Furthermore, these amino acids at high concentrations significantly inhibited the binding of Ni2+from nickel subsulfide to deproteinized DNA from renal cortical cells, whereas such inhibition due to Mg2+was close to significant (0.1 >p> 0.05).In vitroexposures of renal cortical cells to nickel subsulfide (0–2 mM) increased the formation of reactive oxygen species in concentration-dependent manner. Furthermore, coincubation of 2 mM nickel subsulfide with either catalase, dimethylthiourea, mannitol, or vitamin C at 37°C for 2 h resulted in a significant decrease of nickel subsulfide-induced formation of DNA–protein crosslinks, suggesting that nickel subsulfide-induced DNA–protein crosslink formation in isolated rat renal cortical cells is caused by the formation of reactive oxygen species. The potent protective effects of these specific amino acids and Mg2+against nickel subsulfide-induced DNA–protein crosslink formation in isolated renal cortical cells are due to reduction of cellular uptake of Ni2+and inhibition of the binding of Ni2+to deproteinized DNA.

Aldosterone metabolism in cultures of rat renal cortical and medullary cells

The metabolism of 4-14C-d-aldosterone (at 3 nM) was studied in the primary target organ of the hormone, in renal cortical and medullary cell cultures obtained from Wistar rats. Larger amounts of aldosterone were metabolized in medullary cells than in cortical cells, as measured by a decreased 4-14

Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules

Control of calcium oxalate crystal structure and cell adherence by urinary macromolecules

Potential metabolism and cytotoxicity of N-(3,5-dichlorophenyl)succinimide and its hepatic metabolites in isolated rat renal cortical tubule cells

N-(3,5-Dichlorophenyl)succinimide (NDPS) is an agricultural fungicide and antimicrobial agent that produces nephrotoxicity in rats. The contribution of the kidney, if any, to the mechanism of toxicity of NDPS is not known. Therefore, the ability of isolated renal cortical tubule cells to metabolize NDPS and some of its known hepatic metabolites was studied. The cytotoxic potential of these compounds was also assessed. Renal cortical tubule cells were isolated by collagenase digestion and were incubated with the test compounds (2 mM) for 3 h. Metabolite formation was monitored by reversed phase HPLC and cell viability was assessed using trypan blue exclusion. The isolated kidney cells do not appear to metabolize NDPS or any of its known hepatic metabolites. In addition, none of these compounds were directly cytotoxic to the renal cells. However, the cells were susceptible to mercuric chloride (1 mM) and chloroform (125 or 200 mM). Intracellular glutathione levels were unaltered by the presence of NDPS in the incubations. These results suggest that NDPS and its metabolites are not directly toxic to the kidney and are not converted into the ultimate nephrotoxic species by the kidney. Extrarenal metabolism may, therefore, be critical to the expression of NDPS-induced nephrotoxicity.

Apical and basolateral parathyroid hormone receptors in rat renal cortical membranes.

Brush border (BBM) and basolateral membranes (BLM) of rat renal cortical cells separated by free flow electrophoresis revealed two distinct peaks of BBM-specific leucine aminopeptidase and Na+/K(+)-ATPase for BLM. PTH/PTH-related protein (PTHrP) receptors were identified in BBM and BLM. Specific binding of 125 pM [125I]chicken [Tyr36]-PTHrP-(1-36)amide [chPTHrP-(1-36)] to individual fractions of membranes separated by free flow electrophoresis overlapped with the leucine aminopeptidase and Na+/K(+)-ATPase profiles. Binding to pooled BBM was 53 +/- 5% (mean +/- SEM) of that to BLM (P < 0.01). In BBM and BLM, half-maximal inhibition of binding was obtained with 0.4-0.9 nM chPTHrP-(1-36) and 0.2-0.6 nM rat PTH-(1-34). Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM) lowered chPTHrP-(1-36) binding to 50% of control levels, and half-maximal inhibition of binding was obtained with 480 and 8 nM GTP gamma S in BBM and BLM, respectively. Cross-linking of the PTH/PTHrP receptors with [125I]chPTHrP-(1-36) modified with N-hydroxysuccinimidyl-4-azidobenzoate revealed indistinguishable doublets of 83 and 73 kilodaltons in both BBM and BLM. Adenylyl cyclase was stimulated 6- and 10-fold by chPTHrP-(1-36) and GTP gamma S, respectively, in BLM and 1.3- and 1.9-fold in BBM. In conclusion, PTH receptors were recognized in both the basolateral and brush border membranes. Different receptor coupling to G-proteins and minimal cAMP stimulation in BBM provide evidence for PTH/PTHrP receptor isotypes and/or different postreceptor activation in BBM and BLM.

The Effect of Dopamine on Adenylate Cyclase and Na+, K+-ATPase Activity in the Developing Rat Renal Cortical and Medullary Tubule Cells

Dopamine has an age-dependent natriuretic and diuretic effect. We have investigated the ontogeny of the dopamine response on adenylate cyclase activity and Na+,K(+)-ATPase activity in two different cell populations in the infant (10-d-old) and the adult (40-d-old) rat kidney. Basal- and forskolin-stimulated adenylate cyclase activity in tubular suspensions of renal cortex was 5.4-fold (p < 0.05) higher in the 10-d-old rats than in the 40-d-old rats but unchanged between the ages in a suspension of medullary tubules. The dopamine-1-specific agonist fenoldopam did not stimulate adenylate cyclase activity in the cortical cells from 10-d-old rats but did stimulate activity 51 +/- 16% (p < 0.05) in the 40-d-old rats. In the medullary suspension, fenoldopam stimulated adenylate cyclase activity by 43.5 +/- 5% (p < 0.001) in the 10-d-old rats and by 32.0 +/- 7% (p < 0.01) in the 40-d-old rats. In the isolated proximal convoluted tubule, dopamine inhibited Na+,K(+)-ATPase activity in both the 10-d-old (34 +/- 3%, p < 0.001) and 40-d-old rats (44 +/- 7%, p < 0.001). In contrast, in the medullary thick ascending limb of Henle, inhibition of Na+,K(+)-ATPase activity by fenoldopam was more pronounced in the 10-d-old (56 +/- 6%, p < 0.001) than in the 40-d-old rat (33 +/- 6%, p < 0.001). In summary, the renal tubular effects of dopamine on adenylate cyclase and Na+,K(+)-ATPase activity change during postnatal development in a cell-specific manner.

Glutathione depletion, lipid peroxidation, DNA double-strand breaks and the cytotoxicity of 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone in rat renal cortical cells

The mechanisms involved in the cytotoxicity of 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone, a model compound for hydroquinone derived mercapturic acids, were investigated in rat renal proximal tubule cells. 2-Bromo-3-( N-acetylcystein- S-yl)hydroquinone induced a time- and concentration-dependent decrease in cell viability and in the levels of cellular glutathione. Antioxidants such as N,N′-diphenyl- p-phenylene diamine and ascorbic acid and the iron chelator desferrioxamine very efficiently protected the cells from 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone without influencing glutathione depletion. The acetoxymethyl ester of the Ca 2+ chelator Quin-2, the inhibitor of the Ca 2+- and Mg 2+- dependent endonucleases, aurintricarboxylic acid and the poly(ADP-ribose)-polymerase inhibitor 3-aminobenzamide also ameliorated 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone cytotoxicity. Moreover, 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone depleted Ca 2+ from isolated kidney mitochondria, increased the amount of malondialdehyde in rat kidney cells and induced DNA double-strand breaks in renal cells in culture. These results suggest that renal cells oxidize 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone to the corresponding quinone; this soft electrophile reacts rapidly with glutathione, thus depleting cellular glutathione concentrations as indicated by the tentative identification of a 2-bromo-3-( N-acetylcystein- S-yl)hydroquinone thioether in the incubation medium of renal cells treated with the mercapturate. As a result of the massive glutathione depletion, peroxidative mechanisms then cause an elevation of the cytosolic concentrations of ionized calcium; impairment of the ability of the mitochondria to sequester Ca 2+ plays an important role in the elevation of the Ca 2+ concentration. Finally, activation of Ca 2+- and Mg 2+-dependent endonucleases results in DNA damage and cell death.

A biphasic effect of noradrenaline on renin release from rat juxtaglomerular cells in vitro is mediated by alpha 1- and beta-adrenoceptors.

The direct effect of noradrenaline on renin release from juxtaglomerular (JG) cells in vitro were investigated in a dynamic superfusion system of dispersed rat renal cortical cells. At low concentrations (1-100 nmol/l), noradrenaline stimulated renin release in a dose-dependent manner, while at higher concentrations (0.1-1 mmol/l) it inhibited renin release. The stimulatory effect of 0.1 mumol noradrenaline/l was completely blocked by a beta-adrenoceptor antagonist, propranolol (0.1 mumol/l). When applied at concentrations of 1 mumol/l or 10 mumol/l, noradrenaline had no consistent effect on renin release, although 10 mumol noradrenaline/l had an inhibitory effect in the presence of propranolol (0.1 mumol/l). The inhibitory effect of noradrenaline (0.1 mmol/l) was converted to a stimulatory effect by the addition of an alpha 1-adrenoceptor antagonist (bunazosin, 1 mumol/l), but was not altered by the addition of an alpha 2-adrenoceptor antagonist (yohimbine, 1 mumol/l). These results indicate that low concentrations of noradrenaline directly stimulate renin release from JG cells by the activation of beta-adrenoceptors, while high concentrations of noradrenaline inhibit renin release by the activation of alpha 1-adrenoceptors. Accordingly, a dynamic balance may exist between beta-adrenergic stimulation and alpha 1-adrenergic depression of renin release.

Rapid release of bound glucose-6-phosphate dehydrogenase by growth factors. Correlation with increased enzymatic activity

Epidermal growth factor (EGF), a mitogen for renal proximal tubule cells, activated the hexose monophosphate (HMP) shunt in renal proximal tubule cells (Stanton, R. C., and Seifter, J. L. (1988) Am. J. Physiol. 254, C267-C271). We therefore evaluated the effect of EGF on the HMP shunt enzymes glucose 6-phosphate dehydrogenase (G6PD, the rate-limiting enzyme) and 6-phosphogluconate dehydrogenase. Rat renal cortical cells (RCC) were incubated with either EGF or platelet-derived growth factor (PDGF) and then assayed for G6PD and 6-phosphogluconate dehydrogenase activities. EGF and PDGF increased G6PD activity by 25 and 27% respectively. Although phorbol myristate acetate (PMA), ionomycin, PMA + ionomycin, and 8-bromo-cyclic AMP had no significant effect on the activity, a 5-min preincubation with PMA potentiated the activation of G6PD by PDGF. Growth factor activation of G6PD was also seen in a fibroblast and epithelial cell line. None of the agents affected 6-phosphogluconate dehydrogenase activity in the RCC or in the cell lines. Further exploration into a possible mechanism for G6PD activation revealed that growth factors caused release of G6PD from a structural element within the cell. Streptolysin O permeabilization of RCC did not cause significant release of G6PD. However, within 1 min of addition of EGF or PDGF to permeabilized cells, G6PD was released into the cell supernatant. The nonhydrolyzable analog of GTP, guanosine 5'-O-(thiotriphosphate), caused a similar release of G6PD. Preincubation with pertussis toxin or guanyl-5'-yl thiophosphate inhibited the PDGF but not the EGF effect. Although the data do not establish a definitive proof linking G6PD release and G6PD activation, these results suggest that they are related. Thus, growth factor stimulation of the HMP shunt likely occurs by a novel mechanism associated with release of bound G6PD.