Dopamine induces ERK activation in renal epithelial cells through H2O2 produced by monoamine oxidase

BACKGROUND: The rat renal proximal tubule cells contain a large amount of monoamine oxidase, which catalyzes the oxidative deamination of catecholamines such as dopamine (DA). The aim of this study is to investigate the potential role of hydrogen peroxide (H2O2) produced by monoamine oxidase (MAO) isoform on regulation of cell signaling and function. METHODS: Primary rat proximal tubular cells, which contain almost exclusively MAO-A, and human embryonic kidney 293 (HEK 293) cells stably transfected with human MAO-B cDNA were treated with DA or tyramine in the presence or the absence of some inhibitors. Then, Shc protein tyrosine phosphorylation and extracellular-regulated kinase (ERK) activation were evaluated by immunoprecipitation/immunoblot analysis and cell proliferation by [3H]thymidine incorporation or cell counting. RESULTS: In rat proximal tubule cells, DA induced tyrosine phosphorylation of Shc, ERK activation, and a significant increase in DNA synthesis. The involvement of MAO-dependent H2O2 generation induced by DA (5 micromol/L) was supported by the demonstration that the DA effects were (1) fully prevented by cell pretreatment with the MAO inhibitor pargyline, the antioxydant N-acetylcysteine (NAC), and the DA uptake inhibitor GBR 12909; (2) not abrogated by the D1 and D2 receptor antagonists; (3) observed in HEK 293 MAO-B cells but not in HEK 293 wild-type cells, which do not express MAO; and (4) similar to those induced by another MAO substrate, tyramine. CONCLUSIONS: Taken together, these results show that in addition to the effects related to receptor stimulation, DA, and probably the other catecholamines, may induce some of its effects through the MAO-dependent H2O2 production.     

Dopamine acutely decreases apical membrane Na/H exchanger NHE3 protein in mouse renal proximal tubule

BACKGROUND: Dopamine is a principal natriuretic hormone in mammalian Na+ homeostasis. Dopamine acutely alters glomerular filtration rate (GFR) and decreases Na+ absorption in both the proximal and distal nephron. Proximal tubule natriuresis is effected through inhibition of the apical membrane Na/H exchanger NHE3. METHODS: We examined whether dopamine directly and acutely decreases apical membrane NHE3 protein using renal tissue in two in vitro systems: renal cortical slices and in vitro perfused single tubules. After incubation with dopamine, NHE3 activity was measured by 22Na flux and NHE3 antigen was measured by immunoblot in apical membrane and total cellular membranes. RESULTS: Direct application of dopamine to either cortical slices or microperfused tubules acutely decreases NHE3 activity and antigen at the apical membrane of the proximal tubule. No change in total cellular NHE3 was detected. CONCLUSION: One mechanism by which dopamine causes natriuresis is via direct and acute reduction of NHE3 protein at the apical membrane via changes in NHE3 protein trafficking.     

Ginsenosides protect apical transporters of cultured proximal tubule cells from dysfunctions induced by h(2)o(2)

Oxidative stress has been implicated as a primary cause of renal failure in certain renal diseases. Indeed, renal proximal tubule is a very sensitive site to oxidative stress and retains functionally fully characterized transporters. It has been reported that ginsenosides have a beneficial effect on diverse diseases including oxidative stress. However, the protective effect of ginsenosides on oxidative stress has not been elucidated in renal proximal tubule cells. Thus, we examined the effect of ginsenosides on oxidative stress-induced alteration of apical transporters and its related mechanism in renal proximal tubule cells. In the present study, hydrogen peroxide (H(2)O(2)) (>10(-5) M) inhibited alpha-methyl-D-glucopyranoside uptake in a dose-dependent manner (p < 0.05). It also inhibited Pi and Na(+) uptake. At a concentration of 20 microg/ml, total ginsenosides significantly reduced H(2)O(2)-induced inhibition of apical transporters. In contrast, protopanaxadiol (PD) and protopanaxatriol (PT) saponins exhibited a less preventive effect than total ginsenosides (p < 0.05). Furthermore, we examined its action mechanism. H(2)O(2) increased lipid peroxide formation, arachidonic acid (AA) release, and Ca(2+) uptake. These effects on H(2)O(2) were significantly prevented by total ginsenosides and PD or PT sanponins. However, total ginsenosides appear to be more protective than PD and PT saponins (p < 0.05). In conclusion, ginsenosides prevented H(2)O(2)-induced inhibition of apical transporters via a decrease in oxidative stress, AA release, and Ca(2+) uptake in primary cultured renal proximal tubule cells.     

D1 dopamine receptor signaling involves caveolin-2 in HEK-293 cells

BACKGROUND: Dopamine receptors in the kidney, especially those belonging to the D1-like receptor family, are important in the regulation of renal function and blood pressure. Because of increasing evidence that G protein-coupled receptors (GPCRs) are associated with caveolae and lipid rafts, we tested the hypothesis that the D1 dopamine receptor (D1R) and signaling molecules are regulated by caveolin in caveolae or lipid rafts. METHODS: Six experimental approaches were used: (1) construction of tagged human D1Rs (hD1Rs) and transfectants; (2) cell culture [human embryonic kidney (HEK)-293 and immortalized rat renal proximal tubule cells] and biotinylation; (3) cell fractionation by sucrose gradient centrifugation; (4) immunoprecipitation and immunoblotting; (5) immunofluorescence and confocal microscopy; and (6) adenylyl cyclase assays. RESULTS: hD1Rs, heterologously expressed in HEK-293 cells, formed protein species with molecular mass ranging from 50 to 250 kD, and were localized in lipid rafts and nonraft plasma membranes. The hD1Rs cofractionated with caveolin-2, G protein subunits, and several signaling molecules. Both exogenously expressed hD1Rs and endogenously expressed rat D1Rs colocalized and coimmunoprecipitated with caveolin-2. A D1R agonist (fenoldopam) increased the amount of caveolin-2beta associated with hD1Rs and activated adenylyl cyclase to a greater extent in lipid rafts than in nonraft plasma membranes. Reduction in the expression of caveolin-2 with antisense oligonucleotides attenuated the stimulatory effect of fenoldopam on cyclic adenosine monophosphate (cAMP) accumulation. CONCLUSION: The majority of hD1Rs are distributed in lipid rafts. Heterologously and endogenously expressed D1Rs in renal cells are associated with and regulated by caveolin-2.     

Hypoxia/re-oxygenation injury induces apoptosis of LLC-PK1 cells by activation of caspase-2

Hypoxia/re-oxygenation injury induces apoptosis in renal tubule cells, but its underlying molecular pathways are not fully elucidated. Activation of caspase-2 has recently been proposed as a novel mechanism of apoptosis in fibroblasts. In this study we examined whether hypoxia/re-oxygenation injury induces apoptosis in proximal tubule cells by activation of caspase-2. Porcine proximal tubule (LLC-PK1) cells were subjected to hypoxia/re-oxygenation injury in the presence or absence of caspase inhibitors. Apoptosis was detected by DNA laddering, flow cytometry, and immunocytochemistry for Bax and cytochrome c. The activity of caspases-2, 8 and 9 was measured. Apoptosis was evident after hypoxia/re-oxygenation and was best prevented by pretreatment with caspase-2 inhibitor. Hypoxia/re-oxygenation resulted in a dramatic increase in caspase-2 activity (32-fold, in comparison with a 16-fold increase in caspase-8 activity and a tenfold increase in caspase-9 activity). Immunocytochemistry revealed Bax activation and translocation to mitochondria and cytochrome c release into the cytosol following hypoxia/re-oxygenation, both of which were significantly suppressed by pretreatment with caspase-2 inhibitor. These results indicate that hypoxia/re-oxygenation injury in cultured proximal tubule cells induced apoptosis by activation of caspase-2, which is required for the mitochondrial translocation of Bax.     

Human renal organic anion transporter 4 operates as an asymmetric urate transporter

Human organic anion transporter 4 (hOAT4) is located at the apical membrane of proximal tubule cells and involved in renal secretion and reabsorption of endogenous substances as well as many drugs and xenobiotics. This study reevaluated the physiologic role, transport mode, and driving forces of hOAT4. 6-Carboxyfluorescein (6-CF) uptake into HEK293 cells that stably expressed hOAT4 was saturable, resulting in a K(m) of 108 muM. 6-CF as well as [(3)H]estrone sulfate ([(3)H]ES) accumulation by HEK293-hOAT4 cells were abolished by ES, dehydroepiandrosterone sulfate, sulfinpyrazone, benzbromarone, and probenecid, whereas several OA, including p-aminohippurate (PAH), lactate, pyrazinoate, nicotinate, glutarate, and the diuretic hydrochlorothiazide (HCTZ) exhibited a slight or a NS inhibitory effect. PAH and glutarate are not taken up by HEK293-hOAT4 cells, but they trans-stimulated 6-CF and [(3)H]ES uptake, indicating an asymmetric interaction of hOAT4 with these substrates. In chloride-free medium, HEK293-hOAT4-mediated [(3)H]PAH efflux was almost abolished, whereas addition of ES restored it comparable to Ringer solution, consistent with a physiologic ES/PAH or PAH/Cl(-) exchange mode of hOAT4. Moreover, an acidification of the uptake medium increased 6-CF as well as [(3)H]ES uptake, which was reduced by nigericin, suggesting that hOAT4 also can operate as an OA/OH(-) exchanger. hOAT4 facilitates substantial uptake of [(14)C]urate, which was elevated 2.6-fold by intracellular HCTZ. Thus, hOAT4 is the long-postulated, low-affinity apical urate anion exchanger that facilitates HCTZ-associated hyperuricemia.     

Binding of losartan to angiotensin AT1 receptors increases dopamine D1 receptor activation

Signaling through both angiotensin AT1 receptors (AT1R) and dopamine D1 receptors (D1R) modulates renal sodium excretion and arterial BP. AT1R and D1R form heterodimers, but whether treatment with AT1R antagonists functionally modifies D1R via allosterism is unknown. In this study, the AT1R antagonist losartan strengthened the interaction between AT1R and D1R and increased expression of D1R on the plasma membrane in vitro. In rat proximal tubule cells that express endogenous AT1R and D1R, losartan increased cAMP generation. Losartan increased cAMP in HEK 293a cells transfected with both AT1R and D1R, but it did not increase cAMP in cells transfected with either receptor alone, suggesting that losartan induces D1R activation. Furthermore, losartan did not increase cAMP in HEK 293a cells expressing AT1R and mutant S397/S398A D1R, which disrupts the physical interaction between AT1R and D1R. In vivo, administration of a D1R antagonist significantly attenuated the antihypertensive effect of losartan in rats with renal hypertension. Taken together, these data imply that losartan might exert its antihypertensive effect both by inhibiting AT1R signaling and by enhancing D1R signaling.     

Crystals cause acute necrotic cell death in renal proximal tubule cells, but not in collecting tubule cells

BACKGROUND: The interaction between renal tubular cells and crystals generated in the tubular fluid could play an initiating role in the pathophysiology of calcium oxalate nephrolithiasis. Crystals are expected to form in the renal collecting ducts, but not in the proximal tubule. In the present investigation, we studied the damaging effect of calcium oxalate crystals on renal proximal and collecting tubule cells in culture. METHODS: Studies were performed with the renal proximal tubular cell lines, porcine proximal tubular cells (LLC-PK(1)) and Madin-Darby canine kidney II (MDCK-II) and the renal collecting duct cell lines, RCCD(1) and MDCK-I. Confluent monolayers cultured on permeable growth substrates in a two-compartment culture system were apically exposed to calcium oxalate monohydrate crystals, after which several cellular responses were studied, including monolayer morphology (confocal microscopy), transepithelial electrical resistances (TER), prostaglandin E(2) (PGE(2)) secretion, DNA synthesis ([(3)H]-thymidine), total cell numbers, reactive oxygen species [hydrogen peroxide (H(2)O(2))] generation, apoptotic (annexin V and DNA fragmentation), and necrotic (propidium iodide influx) cell death. RESULTS: Crystals were rapidly taken up by proximal tubular cells and induced a biphasic response. Within 24 hours approximately half of the cell-associated crystals were released back into the apical fluid (early response). Over the next 2 weeks half of the remaining internalized crystals were eliminated (late response). The early response was characterized by morphologic disorder, increased synthesis of PGE(2), H(2)O(2), and DNA and the release of crystal-containing cells from the monolayers. These released cells appeared to be necrotic, but not apoptotic cells. Scrape-injured monolayers generated even higher levels of H(2)O(2) than those generated in response to crystals. During the late response, crystals were gradually removed from the monolayers without inflammation-mediated cell death. Crystals did not bind to, were not taken up by, and did not cause marked responses in collecting tubule cells. CONCLUSION: This study shows that calcium oxalate crystals cause acute inflammation-mediated necrotic cell death in renal proximal tubular cells, but not in collecting tubule cells. The crystal-induced generation of reactive oxygen species by renal tubular cells is a general response to tissue damage and the increased levels of DNA synthesis seem to reflect regeneration rather than growth stimulation. As long as the renal collecting ducts are not obstructed with crystals, these results do not support an important role for crystal-induced tissue injury in the pathophysiology of calcium oxalate nephrolithiasis.     

Inhibition of c-Jun N-terminal kinase ameliorates apoptosis induced by hydrogen peroxide in the kidney tubule epithelial cells (NRK-52E)

BACKGROUND: Hydrogen peroxide (H2O2)-induced apoptosis has been shown to be involved in ischemic and toxic tubular damage. Recent studies have revealed that oxidative stress can activate c-Jun N-terminal kinase (JNK), and the oxidative stress-JNK pathway is an important apoptotic pathway of cells exposed to various stresses. The present study was designed to investigate JNK activation and the effects of the JNK pathway inhibition during H2O2-induced apoptosis in kidney epithelial tubule cells (NRK-52E). MATERIALS AND METHODS: NRK-52E cells were treated with 0-500 microM H2O2 and/or 100 microM quercetin (an inhibitor of the JNK pathway). Apoptosis was assessed by flow cytometry analysis and DNA ladder. JNK activity was assessed by the GST-c-Jun (1-169) binding/protein kinase assay. RESULTS: H2O2 induced apoptosis in NRK-52E cells in a concentration-dependent manner, which was demonstrated by the reduced DNA PI staining, externalization of phosphatidylserine and DNA ladder. Apoptosis induced by H2O2 was accompanied by JNK activation and up-regulated JNK activity. Quercetin treatment suppressed the JNK activity and ameliorated apoptosis induced by H2O2. CONCLUSIONS: H2O2 induced apoptosis in NRK-52E cells, which was associated with activation and up-regulation of JNK. Quercetin treatment could decrease JNK activity and ameliorate H2O2-induced apoptosis.     

Stimulation of proximal tubular cell apoptosis by albumin-bound fatty acids mediated by peroxisome proliferator activated receptor-gamma

In nephrotic syndrome, large quantities of albumin enter the kidney tubule. This albumin carries with it a heavy load of fatty acids to which the proximal tubule cells are exposed at high concentration. It is postulated that exposure to fatty acids in this way is injurious to proximal tubule cells. This study has examined the ability of fatty acids to interact with peroxisome proliferator-activated receptors (PPAR) in primary cultures of human proximal tubule cells. Luciferase reporter assays in transiently transfected human proximal tubule cells were used to show that albumin bound fatty acids and other agonists activate PPARgamma in a dose-dependent manner. One of the consequences of this activation is apoptosis of the cells as determined by changes in cell morphology, evidence of PARP cleavage, and appearance of DNA laddering. Overexpression of PPARgamma in these cells also results in enhanced apoptosis. Both fatty acid-induced PPAR activation and apoptosis in these cells can be blocked by PPAR response element decoy oligonucleotides. Activation of PPARgamma by the specific agonist PGJ(2) is associated with inhibition of cell proliferation, whereas activation by albumin bound fatty acids is accompanied by increased proliferation. However, the net balance of apoptosis/proliferation favors deletion of cells. These results implicate albumin-bound fatty acids as important mediators of tubular injury in nephrosis and provide fresh impetus for pursuit of lipid-lowering strategies in proteinuric renal disease.     

Ontogeny of aromatic L-amino acid decarboxylase-containing tubule cells in rat kidney.

Dopamine plays an important role in regulation of renal sodium transport. Proximal tubule cells produce dopamine after decarboxylation of L-DOPA via the enzyme aromatic L-amino acid decarboxylase (AADC). The presence and cellular localization of AADC-like immunoreactivity (-LI) and AADC mRNA were examined during pre- and postnatal development in rat kidney by indirect immunofluorescence and in situ hybridization histochemistry. Few scattered condensations of AADC-immunoreactive (-IR) tubule cells forming a lumen were detected on gestational day 18. From gestational day 21, many AADC-IR tubule cells were observed in the inner cortex, whereas the outer cortex lacked AADC-LI. Within 24 hours of birth, AADC-IR cells in the inner cortex could be identified as proximal tubule cells. During day 3 and 5 there was an increase in number of AADC-IR proximal tubule cells in the inner cortex, leaving less amount of AADC-negative cells in the outer cortex. Starting from day 8, AADC-IR cells could be seen in the outer cortex. An apparent decrease in AADC-immunofluorescence intensity was observed at 40 days, and at 80 days virtually no AADC-LI could be demonstrated. Intrarenal levels of dopamine showed a tendency to increase between 3 and 20 days, and showed significant decreases between 20 to 40 days and between 40 to 80 days. AADC mRNA was not detected in the kidney at 18 hours after birth, but could be observed in the inner cortex at 6 days. At 12, 19 and 40 days AADC mRNA was seen in the entire cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of sphingosine 1-phosphate receptor 2 protects against renal ischemia-reperfusion injury

Activation of the sphingosine 1-phosphate receptor 1 (S1P(1)R) protects against renal ischemia-reperfusion (IR) injury and inflammation, but the role of other members of this receptor family in modulating renal IR injury is unknown. We found that a selective S1P(2)R antagonist protected against renal IR injury in a dose-dependent manner. Consistent with this observation, both S1P(2)R-deficient mice and wild-type mice treated with S1P(2)R small interfering RNA had reduced renal injury after IR. In contrast, a selective S1P(2)R agonist exacerbated renal IR injury. The S1P(2)R antagonist increased sphingosine kinase-1 (SK1) expression via Rho kinase signaling in renal proximal tubules; the S1P(2)R agonist decreased SK1. S1P(2)R antagonism failed to protect the kidneys of SK1-deficient mice or wild-type mice pretreated with an SK1 inhibitor or an S1P(1)R antagonist, suggesting that the renoprotection conferred by S1P(2)R antagonism results from pathways involving activation of S1P(1)R by SK1. In cultured human proximal tubule (HK-2) cells, the S1P(2)R antagonist selectively upregulated SK1 and attenuated both H(2)O(2)-induced necrosis and TNF-α/cycloheximide-induced apoptosis; the S1P(2)R agonist had the opposite effects. In addition, increased nuclear hypoxia inducible factor-1α was critical in mediating the renoprotective effects of S1P(2)R inhibition. Finally, induction of SK1 and S1P(2)R in response to renal IR and S1P(2)R antagonism occurred selectively in renal proximal tubule cells but not in renal endothelial cells. Taken together, these data suggest that S1P(2)R may be a therapeutic target to attenuate the effects of renal IR injury.     

Desensitization of human renal D1 dopamine receptors by G protein-coupled receptor kinase 4

BACKGROUND: The D1 dopamine receptor, expressed in several nephron segments, participates in the regulation of water and electrolyte transport. Because the renal D1 receptor is desensitized in genetic hypertension, we sought to determine the mechanism(s) of the desensitization of D1 receptors endogenously expressed in renal proximal tubules. METHODS: The mechanisms involved in the homologous desensitization of the D1 receptor in human renal proximal tubule cells were studied by measuring the production of cAMP in response to stimulation or inhibition of G protein-coupled receptor kinase (GRK) activity and expression. Protein expression was assessed by immunoblotting. RESULTS: In human renal proximal tubule cells, the D1 agonist, fenoldopam, increased cAMP accumulation (73 +/- 2%). Fenoldopam pre-treatment decreased the responsiveness to subsequent fenoldopam stimulation (t(1/2) approximately equal to 20 min) with complete desensitization at 30 minutes. Recovery occurred gradually (t(1/2) approximately equal to 20 min) with full recovery at 60 minutes. Forskolin pretreatment minimally affected the fenoldopam effect, indicating a minor involvement of protein kinase A in the homologous desensitization process. Because GRKs are involved in the homologous desensitization process, we determined the consequences of inhibition of GRK expression and activity. Heparin, an inhibitor of GRK activity, decreased the expression of GRK2 and GRK4 and attenuated the desensitization of the D1 receptor (85 +/- 1%). Antisense oligonucleotides (GRK4> GRK2) blunted the D1 receptor desensitization. However, the first 20 minutes of homologous desensitization were not affected by either heparin or GRK antisense oligonucleotides. CONCLUSION: These studies document the critical role of GRK4, relative to GRK2, in the homologous desensitization of D1 receptors in renal proximal tubule cells. However, the early phase of homologous desensitization is regulated by a non-GRK-mediated pathway.     

Renal tubule necrosis and apoptosis modulation by A1 adenosine receptor expression

We have shown that A1 adenosine receptors (A1ARs) are cytoprotective against renal tubular necrosis and apoptosis both in vivo and in vitro. To study the role of A1AR numbers on renal epithelial cell survival, we stably overexpressed the human A1 receptor in a porcine renal tubule cell line and utilized primary cultures of proximal tubules obtained from A1AR knockout mice. Receptor-overexpressing cells were protected against peroxide-induced necrosis and tumor necrosis factor-alpha/cycloheximide-induced apoptosis. Conversely, cultured proximal tubule cells from receptor knockout mice showed more necrotic and apoptotic cell loss than corresponding cells from wild-type mice. Overexpression of the receptor resulted in a significantly higher baseline expression of both total and phosphorylated heat-shock protein (HSP)27; the latter due to A1 receptor enhancement of p38 and AP2 mitogen-activated protein kinase activities. The resistance to cell death in the porcine cells was reversed by selective A1 receptor antagonism and by a selective inhibitor of HSP synthesis. Receptor activation in wild-type mice in vivo led to increased total and phosphorylated HSP27, whereas receptor knockout mice showed decreased baseline and adenosine-mediated HSP phosphorylation. These studies show that endogenous A1AR activation produces cytoprotective effects in renal proximal tubules by modulating HSP27 signaling pathways.     

Free reactive oxygen species and nephrotoxicity of contrast agents

BACKGROUND: The nephrotoxicity induced by contrast media remains a serious clinical problem, and the underlying mechanism has not been completely understood. Experimental and clinical investigations suggest that reactive oxygen species (ROS) are critical determinants of radiocontrast nephropathy (RCN), and that antioxidants can prevent this damage. METHODS: Cultured human proximal renal tubule cells (HK-2) were exposed to hydrogen peroxide (H2O2) at different concentrations. H2O2-induced tubular DNA damage was examined in the presence of the antioxidant MESNA (sodium-2-mercaptoethane sulphonate). The induction of DNA damage was measured with the alkaline comet assay (single cell gel electrophoresis). We also studied 12 patients with stable renal impairment (median baseline creatinine 296 micromol/l; range: 203-495 micromol/l) undergoing cardiac catheterization/intervention prospectively. Patients received 800 mg MESNA intravenously 30 min before exposure to the contrast agent in addition to 0.9% saline hydration. RESULTS: In the cell cultures, oxidative stress on HK-2 cells induced increased DNA migration in the comet assay. Treatment of tubular cells with the antioxidant MESNA prior to the addition of H2O2 significantly reduced DNA migration in the comet assay. In the clinical study, treatment of the patients with MESNA prevented the adverse renal effect of contrast media (median serum creatinine 293; range: 187-433 micromol/l) 48 h after coronary angiography/intervention. CONCLUSION: Both the in vivo and the in vitro studies suggest that the ROS-mediated renal injury could be inhibited by a potent antioxidant such as MESNA.     

Death-associated protein kinase localization to human renal tubule cells, and increased expression of chronic obstructive uropathy in rats

BACKGROUND: Death-associated protein kinase (DAP kinase) is a Ca2+/calmodulin-dependent serine/threonine kinase implicated as a positive apoptosis mediator. However, little is known about DAP kinase involvement with apoptosis in renal diseases. METHODS: In order to determine whether DAP kinase has a role in renal cell apoptosis in kidney diseases, we performed an immunohistochemical study using a monoclonal antibody to DAP kinase. Firstly, by examining the cellular distribution of DAP kinase in normal human renal tissues and cultured proximal tubule cells. We then used western blotting and immunohistochemical analysis to examine directly whether DAP kinase protein levels could be modulated in rat kidneys with chronic obstructive uropathy created by unilateral ureteric ligation. RESULTS: Immunohistochemistry of normal human kidney tissues showed that DAP kinase was exclusively localized in the cytoplasm of renal tubule cells. Expression analysis of DAP kinase using cultured cells confirmed DAP kinase mRNA and protein presence in human renal tubule cells. Immunocytochemical analysis directly visualized DAP kinase in the cytoplasm of the renal tubule cells in culture. Finally, DAP kinase was found up-regulated in renal tubule cells of rat kidneys with chronic obstructive uropathy. CONCLUSIONS: Our study demonstrates that DAP kinase is localized to renal tubule cells, implying a crucial role for DAP kinase in renal tubular cell apoptosis in progressive renal diseases.     

Mesna or cysteine prevents chloroacetaldehyde-induced cell death of human proximal tubule cells

Chloroacetaldehyde (CAA) is formed in the body from the chemotherapeutically used drug ifosfamide (IFO). CAA leads to cell death in proximal tubule cells mainly through the mechanism of necrosis rather than apoptosis. During chemotherapy, 2-mercaptosulfonic acid (mesna) is used with IFO to protect the urothel from cell damage. Little is known of the effect of mesna on renal proximal tubule cells, the primary site of damage after IFO treatment. Mesna contains a sulfhydryl (SH) group. To clarify whether SH-group-containing molecules can prevent CAA-induced cell death, we studied the effect of mesna and cysteine on necrosis, apoptosis, and protein content in a human proximal tubule-derived cell line (IHKE cells) treated with CAA. Both substances prevented CAA-induced necrotic cell death and protein loss and restored CAA-inhibited caspase-3 activity. CAA also prevented cisplatin-induced apoptosis. This inhibition was reversible in the presence of glutathione (GSH). We conclude that SH-containing molecules can protect proximal tubule cells from cell death because they interact with CAA before CAA can disturb other important cellular SH groups. A sufficient supply of intra- and extracellular SH groups during IFO chemotherapy may therefore have the ability to protect renal tubule cells from cell death.     

Uptake of chemically reactive, DNA-damaging sulfuric acid esters into renal cells by human organic anion transporters

The procarcinogen 1-methylpyrene is activated by hepatic enzymes via 1-hydroxymethylpyrene to 1-sulfooxymethylpyrene (1-SMP), a highly reactive and mutagenic metabolite. Previously, high levels of 1-SMP DNA adducts were observed in rat kidneys after intraperitoneal administration of 1-hydroxymethylpyrene or 1-SMP. This study examined whether organic anion transporters (OAT) that are expressed at the basolateral membrane of proximal tubule cells are involved in uptake of SMP. Human epithelial kidney (HEK293) cells that stably express human OAT1 (hOAT1) and hOAT3 were used. Stable isomers of 1-SMP, (2-SMP and 4-SMP) competitively inhibited the uptake of characteristic substrates p-aminohippurate for hOAT1 and estrone sulfate for hOAT3. Both inhibitors exhibited high affinity for hOAT1 (K(i) = 4.4 microM for 2-SMP; K(i) = 5.1 microM for 4-SMP) as well as hOAT3 (K(i) = 1.9 microM for 2-SMP; K(i) = 2.1 microM for 4-SMP). The uptake rate of 4-SMP (at a concentration of 10 microM) by hOAT1- and hOAT3-expressing cells was 3.0 and 1.6 times higher, respectively, than in control cells. Uptake of the reactive isomer 1-SMP was investigated using as the end point the level of DNA adducts that were formed in the cells. After exposure to 1-SMP (10 microM), the DNA adduct level was 4.6 and 3.0 times higher in hOAT1- and hOAT3-expressing cells, respectively, than in control cells. The enhanced DNA adduct formation in hOAT-expressing cells was abolished in the presence of the OAT inhibitor probenecid. This study indicates that OAT can mediate the basolateral uptake of reactive sulfuric acid esters into proximal tubule cells and thereby participate in kidney cell damage by these compounds.