Localizations of Na(+)-D-glucose cotransporters SGLT1 and SGLT2 in human kidney and of SGLT1 in human small intestine, liver, lung, and heart.

Novel affinity-purified antibodies against human SGLT1 (hSGLT1) and SGLT2 (hSGLT2) were used to localize hSGLT2 in human kidney and hSGLT1 in human kidney, small intestine, liver, lung, and heart. The renal locations of both transporters largely resembled those in rats and mice; hSGLT2 and SGLT1 were localized to the brush border membrane (BBM) of proximal tubule S1/S2 and S3 segments, respectively. Different to rodents, the renal expression of hSGLT1 was absent in thick ascending limb of Henle (TALH) and macula densa, and the expression of both hSGLTs was sex-independent. In small intestinal enterocytes, hSGLT1 was localized to the BBM and subapical vesicles. Performing double labeling with glucagon-like peptide 1 (GLP-1) or glucose-dependent insulinotropic peptide (GIP), hSGLT1 was localized to GLP-1-secreting L cells and GIP-secreting K cells as has been shown in mice. In liver, hSGLT1 was localized to biliary duct cells as has been shown in rats. In lung, hSGLT1 was localized to alveolar epithelial type 2 cells and to bronchiolar Clara cells. Expression of hSGLT1 in Clara cells was verified by double labeling with the Clara cell secretory protein CC10. Double labeling of human heart with aquaporin 1 immunolocalized the hSGLT1 protein in heart capillaries rather than in previously assumed myocyte sarcolemma. The newly identified locations of hSGLT1 implicate several extra renal functions of this transporter, such as fluid absorption in the lung, energy supply to Clara cells, regulation of enteroendocrine cells secretion, and release of glucose from heart capillaries. These functions may be blocked by reversible SGLT1 inhibitors which are under development.

Regulation of Expression of Renal Organic Anion Transporters OAT1 and OAT3 in a Model of Ischemia/Reperfusion Injury.

BACKGROUND: Recently, we gained evidence that impairment of rOat1 and rOat3 expression induced by ischemic acute kidney injury (AKI) is mediated by COX metabolites and this suppression might be critically involved in renal damage. METHODS: (i) Basolateral organic anion uptake into proximal tubular cells after model ischemia and reperfusion (I/R) was investigated by fluorescein uptake. The putative promoter sequences from hOAT1 (SLC22A6) and hOAT3 (SCL22A8) were cloned into a reporter plasmid, transfected into HEK cells and (ii) transcriptional activity was determined after model ischemia and reperfusion as a SEAP reporter gen assay. Inhibitors or antagonists were applied with the beginning of reperfusion. RESULTS: By using inhibitors of PKA (H89) and PLC (U73122), antagonists of E prostanoid receptor type 2 (AH6809) and type 4 (L161,982), we gained evidence that I/R induced down regulation of organic anion transport is mediated by COX1 metabolites via E prostanoid receptor type 4. The latter signaling was confirmed by application of butaprost (EP2 agonist) or TCS2510 (EP4 agonist) to control cells. In brief, the latter signaling was verified for the transcriptional activity in the reporter gen assay established. Therein, selective inhibitors for COX1 (SC58125) and COX2 (SC560) were also applied. CONCLUSION: Our data show (a) that COX1 metabolites are involved in the regulation of renal organic anion transport(ers) after I/R via the EP4 receptor and (b) that this is due to transcriptional regulation of the respective transporters. As the promoter sequences cloned were of human origin and expressed in a human renal epithelial cell line we (c) hypothesize that the regulatory mechanisms described after I/R is meaningful for humans as well.

The nephrotoxic Ifosfamide-metabolite chloroacetaldehyde interferes with renal extracellular matrix homeostasis.

BACKGROUND/AIMS: Chronic renal proximal tubule dysfunction after therapy with the antineoplastic agent ifosfamide (IFO) is often attributed to the metabolite chloroacetaldehyde (CAA). Chronic IFO-nephropathy is reported to result in tubulointerstitial fibrosis and inflammation. METHODS: To elucidate possible effects of CAA on extracellular matrix homeostasis, we investigated the action of CAA on markers of extracellular matrix (ECM) homeostasis in human proximal tubule cells (RPTEC) by use of direct ELISA for extracellular collagens and gelatin zymography. RESULTS: An increase in type III collagen and a decrease in type IV collagen abundance in the media of RPTEC could be observed after exposure to CAA in clinically relevant concentrations. CAA increased intracellular type III and decreased intracellular type IV collagen. MMP-2 activity was decreased but MMP-9 activity unchanged. The enhanced CAA-induced collagen III formation could be attenuated by the intracellular Ca(2+)-chelator BAPTA-AM, the PKA-antagonist H-89 and by extracellular acidification. CAA-induced collagen III abundance was enhanced by db-cAMP and IBMX and by protein overload. CONCLUSIONS: CAA exerts profibrotic effects on RPTEC dependent on Ca(2+) and cAMP/PKA-signaling. These effects are enhanced by additional protein burden and attenuated by acidification. © 2014 S. Karger AG, Basel.

Differential effect of COX1 and COX2 inhibitors on renal outcomes following ischemic acute kidney injury.

BACKGROUND/AIMS: We have previously shown that 1 mg/kg indomethacin improves expression and functionality of renal organic anion transporters Oat1 and Oat3 after renal ischemia and furthermore improves renal outcome after ischemia. As we detected differential effects of COX1 or COX2 inhibitors on organic anion transport after ischemia and reperfusion in culture, we investigated the effect of the SC560 (COX1 inhibitor) and SC58125 (COX2 inhibitor) on expression of Oat1/3 and renal outcome after ischemic acute kidney injury (iAKI). METHODS: iAKI was induced in rats by bilateral clamping of renal arteries for 45 min. SC560 or SC58125 (1 mg/kg each) were given intraperitoneally as soon as reperfusion started. Sham-treated animals served as controls. Oat1/3 were determined by qPCR and Western blot. Glomerular filtration rate (GFR), p-aminohippurate (PAH) clearance and PAH extraction ratio was determined. All parameters were detected 24 h after ischemia. Renal plasma flow was calculated. RESULTS: In clamped animals SC560 (COX1 inhibitor) restored expression of Oat1/3, as well as renal perfusion. Additionally, SC560 substantially improved kidney function as measured by GFR. Application of the COX2 inhibitor SC58125 did not exert these beneficial effects. CONCLUSION: Our study indicates that COX1 inhibitor SC560 applied after ischemia prevents ischemia-induced downregulation of Oat1/3 during reperfusion and has a substantial protective effect on kidney function. Whether and to what particular extent this apparent improvement of function is mechanistically due to beneficial effects on tubular function, renal perfusion or glomerular filtration will be the scope of future studies.

Expression of Na+-D-glucose cotransporter SGLT2 in rodents is kidney-specific and exhibits sex and species differences.

With a novel antibody against the rat Na(+)-D-glucose cotransporter SGLT2 (rSGLT2-Ab), which does not cross-react with rSGLT1 or rSGLT3, the ∼75-kDa rSGLT2 protein was localized to the brush-border membrane (BBM) of the renal proximal tubule S1 and S2 segments (S1 > S2) with female-dominant expression in adult rats, whereas rSglt2 mRNA expression was similar in both sexes. Castration of adult males increased the abundance of rSGLT2 protein; this increase was further enhanced by estradiol and prevented by testosterone treatment. In the renal BBM vesicles, the rSGLT1-independent uptake of [(14)C]-α-methyl-D-glucopyranoside was similar in females and males, suggesting functional contribution of another Na(+)-D-glucose cotransporter to glucose reabsorption. Since immunoreactivity of rSGLT2-Ab could not be detected with certainty in rat extrarenal organs, the SGLT2 protein was immunocharacterized with the same antibody in wild-type (WT) mice, with SGLT2-deficient (Sglt2 knockout) mice as negative control. In WT mice, renal localization of mSGLT2 protein was similar to that in rats, whereas in extrarenal organs neither mSGLT2 protein nor mSglt2 mRNA expression was detected. At variance to the findings in rats, the abundance of mSGLT2 protein in the mouse kidneys was male dominant, whereas the expression of mSglt2 mRNA was female dominant. Our results indicate that in rodents the expression of SGLT2 is kidney-specific and point to distinct sex and species differences in SGLT2 protein expression that cannot be explained by differences in mRNA.

Hypoxia-induced extracellular acidosis increases p-glycoprotein activity and chemoresistance in tumors in vivo via p38 signaling pathway.

Due to inadequate perfusion, tumors develop hypoxia and extracellular acidosis. In vitro, this acidic environment (pH=6.6) has a strong impact on the activity of the p-glycoprotein (pGP) drug transporter responsible for multidrug resistance. This effect is most probably mediated via p38 and/or ERK1/2 signalling pathways. The aim of the study was to analyze whether these findings also play a role for chemosensitivity in solid growing tumors in vivo. Therefore, experimental R3327-AT1 tumors of the rat were exposed to an acidifying treatment leading to forced glycolysis. The intratumoral pO(2)1 was determined polarographically and the extracellular pH was measured with needle electrodes. In addition the cytotoxicity of daunorubicin chemotherapy was assessed. Tumor oxygenation was markedly worsened by the acidosis treatment leading to a mean tumor pO(2) of 1 mmHg. This hypoxia resulted in a pronounced acidification of the tumor tissue from pH 7.04 down to 6.65. Extracellular acidosis in vivo decreased caspase 3-activity after daunorubicin treatment by 30%indicating a reduced chemosensitivity. Inhibition of the p38 signalling pathway restored the reduced chemosensitivity at least partially. However, in contrast to the in vitro findings inhibition of ERK1/2 had practically no impact in vivo.

Implementation of an in vitro model system for investigation of reperfusion damage after renal ischemia.

Ischemic acute kidney injury (iAKI) is a common event in organ transplantation and may occur during severe surgery. To gain mechanistic insights into ischemia-induced alterations at the level of proximal tubule cells we set up an in vitro model of ischemia and reperfusion using the rat proximal tubule cell line NRK-52E. In this particular model we simultaneously applied acidosis, hypoxia and aglycemia together for 2h, using low volume buffer systems and a hypoxia chamber. Thereafter reperfusion was mimicked by subsequently culturing the cells for up to 48h under standard conditions. In order to validate the system we investigated whether effects that take place in existing in vivo models of ischemia and reperfusion can be observed. Namely, induction of necrosis, apoptosis and of ischemia reperfusion induced protein (IRIP), dedifferentiation (alphaSMA), inflammation (MCP-1), inducible NO-synthase (iNOS), release of PGE(2) and basolateral uptake of organic anions. In fact, all parameters developed as described for the in vivo situation during reperfusion after ischemia. Taken altogether we have established an in vitro model of proximal tubule cell reperfusion damage after ischemia, showing typical changes described in vivo. Additionally, our model system is suitable for isolated application of the typical insults associated with ischemia (e.g. acidosis alone, hypoxia alone, aglycemia alone), in order to obtain more insight into the mechanistic events that lead to reperfusion damage in the kidney on the cellular level.

Acidosis induces multi-drug resistance in rat prostate cancer cells (AT1) in vitro and in vivo by increasing the activity of the p-glycoprotein via activation of p38.

Because solid growing tumors often show hypoxia and pronounced extracellular acidosis, the aim of this study was to analyze the impact of an acidotic environment on the activity of the p-glycoprotein (pGP) and on the cellular content and cytotoxicity of the chemotherapeutic drug daunorubicin in the AT1 R-3327 Dunning prostate carcinoma cell line cultured in vitro and in vivo. In vitro, extracellular acidosis (pH 6.6) activated p38 and ERK1/2 and thereby induced daunorubicin resistance via a pronounced activation of pGP. De-novo protein synthesis was not necessary and analysis of transport kinetics indicated a fast and persistent pGP activation at pH 6.6 (when compared with 7.4). Intracellular acidification also induced daunorubicin resistance via activation of pGP, which was mediated by activation of p38 alone. In vivo, tumors were implanted subcutaneously, and the tumor pH was artificially lowered by forcing anaerobic metabolism. In vivo, the reduced extracellular pH of 6.6 was also able to induce daunorubicin resistance, which was abolished by inhibition of p38. These results suggest that pGP activity is dependent on extracellular pH in vitro and in vivo. Moreover, there is strong indication that this effect is mediated via activation of p38 in vivo. Activation of ERK is also suitable to induce pGP activity. Therefore, inhibition of p38 (and ERK) may be used to prevent acidosis induced increase in pGP activity and thereby attenuate multidrug resistance. In addition, supportive treatments reducing tumor acidosis may improve the cytotoxic effect of chemotherapeutic drugs.

Long-term effects of ochratoxin A on fibrosis and cell death in human proximal tubule or fibroblast cells in primary culture.

Ochratoxin A (OTA) is a mycotoxin produced by several fungi which grow on human food source material. Consumption of OTA is almost unavoidable. The consumption leads to low but detectable amounts of OTA in human blood. Risk assessment of OTA is based on studies performed either in animals or cultured cells. So far, mainly cell lines of different origin were used. To be as close as possible to the situation in humans with respect to the experimental setup, we studied the effect of OTA in human proximal tubule cells (RPTEC) and human fibroblasts in primary culture. OTA was administered at concentrations ranging from 0.3 nmol/l up to 10 micromol/l for time periods up to 14 days. Apoptotic and necrotic cell death, collagen I, III, IV and fibronectin secretion as well as NF-kappaB activation were studied. Under our experimental conditions OTA exerted comparable effects on caspase-3 activity and necrosis in both cell types, however RPTEC were more sensitive (order of 10). Surprisingly, very low concentrations of OTA (0.3-10nM) led to cell hypertrophy during prolonged exposure (14 days). RPTEC but not fibroblasts responded with an increase of NF-kappaB activity and collagen III as well as fibronectin secretion underlining the profibrotic action of OTA in the kidney. Collagen I and IV secretion was only slightly changed. The results presented here give good reasons to re-asses the risk of OTA consumption leading to low blood concentrations which have so far been considered harmless.

Ochratoxin A at nanomolar concentrations: a signal modulator in renal cells.

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with nephrotoxic, carcinogenic, and apoptotic potential. Toxicokinetics make the kidney the primary target organ for OTA. Due to its widespread occurrence in improperly stored foodstuff the complete and safe avoidance of OTA for humans is impossible. There are several reports showing a significant correlation between OTA exposure and certain forms of nephropathies. At nanomolar concentrations OTA leads to specific changes of function and phenotype in renal cells. The toxin interacts with certain cellular "key-molecules" (e. g., mitogen-activated protein (MAP) kinases, Ca2+), thereby disturbing cellular signalling and regulation events as well as mitochondrial function. Moreover, OTA has the ability to modulate physiological signals (e. g., angiotensin II or TNFalpha) and thereby influences cell function and cell growth and may even stable re-program the cells (e. g., altered distribution of chromosomes). This review concentrates on the effects of OTA in the nanomolar range and its interactions with cellular signalling networks in different renal cells proposing OTA to act as a signal modulator.

Exposure to nephrotoxic ochratoxin A enhances collagen secretion in human renal proximal tubular cells.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin. There is evidence that OTA leads to cortical interstitial nephropathies in humans, associated with fibrosis. No data are available on the effect of OTA-induced collagen secretion from renal cortical cells. As kidney cortex mainly consists of proximal tubules, we investigated the effect of OTA on particular collagens (I, III, IV) in a well-established proximal tubular cell line (opossum kidney (OK) cells) and in primary cultured human renal proximal tubular epithelial cells (RPTECs). In fibroblasts, OTA neither exerted toxic effects nor induced collagen secretion, most probably due to the absence of suitable uptake mechanisms. OTA exerted time- and dose-dependent toxicity in both OK cells and human RPTECs. Moreover, OTA induced collagen secretion in a time- and dose-dependent manner in both cell types. In opposite to transforming growth factor beta1 (TGF-beta1), OTA incubation induced increased apical secretion of the basement membrane collagen IV. This might be evidence for a loss of cellular polarity after OTA incubation. We conclude that in proximal tubular cells, OTA is able to induce extracellular matrix deposition. As collagen secretion was also inducible in primary cultured human RPTECs, we hypothesize that OTA-induced extracellular matrix deposition by proximal tubular cells may be of importance in generation of renal diseases in humans which are under suspicion of being induced by OTA.

PAH clearance after renal ischemia and reperfusion is a function of impaired expression of basolateral Oat1 and Oat3.

Determination of renal plasma flow (RPF) by para-aminohippurate (PAH) clearance leads to gross underestimation of this respective parameter due to impaired renal extraction of PAH after renal ischemia and reperfusion injury. However, no mechanistic explanation for this phenomenon is available. Based on our own previous studies we hypothesized that this may be due to impairment of expression of the basolateral rate limiting organic anion transporters Oat1 and Oat3. Thus, we investigated this phenomenon in a rat model of renal ischemia and reperfusion by determining PAH clearance, PAH extraction, PAH net secretion, and the expression of rOat1 and rOat3. PAH extraction was seriously impaired after ischemia and reperfusion which led to a threefold underestimation of RPF when PAH extraction ratio was not considered. PAH extraction directly correlated with the expression of basolateral Oat1 and Oat3. Tubular PAH secretion directly correlated with PAH extraction. Consequently, our data offer an explanation for impaired renal PAH extraction by reduced expression of the rate limiting basolateral organic anion transporters Oat1 and Oat3. Moreover, we show that determination of PAH net secretion is suitable to correct PAH clearance for impaired extraction after ischemia and reperfusion in order to get valid results for RPF.

Increased symmetrical dimethylarginine in ischemic acute kidney injury as a causative factor of renal L-arginine deficiency.

Availability of L-arginine, the exclusive substrate for nitric oxide synthases, plays an important role in kidney ischemia/reperfusion injury. The endogenous L-arginine derivatives asymmetrical dimethylarginine (ADMA) and symmetrical dimethylarginine (SDMA) block cellular L-arginine uptake competitively, thereby inhibiting the production of nitric oxide. ADMA also blocks nitric oxide synthase activity directly. Here, we investigate the pathomechanistic impact of ADMA and SDMA on ischemic acute kidney injury. Rats were subject to bilateral renal ischemia (60 minutes)/reperfusion (24 hours) injury. Impairment of renal function was determined with inulin clearance (glomerular filtration rate) and para-aminohippurate (PAH) clearance (renal plasma flow). L-arginine, ADMA, and SDMA levels were measured by liquid chromatography-tandem mass spectrometry. L-arginine was extracted from renal tissue and analyzed by enzyme-linked immunosorbent assay, and protein and messenger RNA expressions were determined by Western blot and real-time reverse transcription polymerase chain reaction. Renal function deteriorated severely after ischemia/reperfusion injury, as demonstrated by inulin and PAH clearance. Serum ADMA and SDMA increased, but tissue expression of specific ADMA or SDMA synthesizing and metabolizing enzymes (protein arginine methyltransferases and dimethyl arginine dimethylaminohydrolases) did not alter. Serum L-arginine increased as well, whereas intracellular L-arginine concentration diminished. Renal messenger RNA expression of cationic amino acid transporters, which mediate L-arginine uptake, remained unchanged. In serum, the ratio of L-arginine to ADMA did not alter after ischemia/reperfusion injury, whereas the ratios of L-arginine to SDMA and ADMA to SDMA decreased. A marked increase in serum SDMA, especially when accompanied by a diminished L-arginine-to-SDMA ratio, might reflect competitive inhibition of cellular L-arginine uptake by SDMA. As a consequence, a pathologic renal L-arginine deficiency in ischemic acute kidney injury results.

Rosiglitazone affects nitric oxide synthases and improves renal outcome in a rat model of severe ischemia/reperfusion injury.

Background. Nitric oxide (NO)-signal transduction plays an important role in renal ischemia/reperfusion (I/R) injury. NO produced by endothelial NO-synthase (eNOS) has protective functions whereas NO from inducible NO-synthase (iNOS) induces impairment. Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor (PPAR)-γ agonist exerted beneficial effects after renal I/R injury, so we investigated whether this might be causally linked with NOS imbalance. Methods. RGZ (5 mg/kg) was administered i.p. to SD-rats (f) subjected to bilateral renal ischemia (60 min). Following 24 h of reperfusion, inulin- and PAH-clearance as well as PAH-net secretion were determined. Morphological alterations were graded by histopathological scoring. Plasma NO(x)-production was measured. eNOS and iNOS expression was analyzed by qPCR. Cleaved caspase 3 (CC3) was determined as an apoptosis indicator and ED1 as a marker of macrophage infiltration in renal tissue. Results. RGZ improves renal function after renal I/R injury (PAH-/inulin-clearance, PAH-net secretion) and reduces histomorphological injury. Additionally, RGZ reduces NO(x) plasma levels, ED-1 positive cell infiltration and CC3 expression. iNOS-mRNA is reduced whereas eNOS-mRNA is increased by RGZ. Conclusion. RGZ has protective properties after severe renal I/R injury. Alterations of the NO pathway regarding eNOS and iNOS could be an explanation of the underlying mechanism of RGZ protection in renal I/R injury.

Acidic environment leads to ROS-induced MAPK signaling in cancer cells.

Tumor micromilieu often shows pronounced acidosis forcing cells to adapt their phenotype towards enhanced tumorigenesis induced by altered cellular signalling and transcriptional regulation. In the presents study mechanisms and potential consequences of the crosstalk between extra- and intracellular pH (pH(e), pH(i)) and mitogen-activated-protein-kinases (ERK1/2, p38) was analyzed. Data were obtained mainly in AT1 R-3327 prostate carcinoma cells, but the principle importance was confirmed in 5 other cell types. Extracellular acidosis leads to a rapid and sustained decrease of pH(i) in parallel to p38 phosphorylation in all cell types and to ERK1/2 phosphorylation in 3 of 6 cell types. Furthermore, p38 phosphorylation was elicited by sole intracellular lactacidosis at normal pH(e). Inhibition of ERK1/2 phosphorylation during acidosis led to necrotic cell death. No evidence for the involvement of the kinases c-SRC, PKC, PKA, PI3K or EGFR nor changes in cell volume in acidosis-induced MAPK activation was obtained. However, our data reveal that acidosis enhances the formation of reactive oxygen species (ROS), probably originating from mitochondria, which subsequently trigger MAPK phosphorylation. Scavenging of ROS prevented acidosis-induced MAPK phosphorylation whereas addition of H(2)O(2) enhanced it. Finally, acidosis increased phosphorylation of the transcription factor CREB via p38, leading to increased transcriptional activity of a CRE-reporter even 24 h after switching the cells back to a normal environmental milieu. Thus, an acidic tumor microenvironment can induce a longer lasting p38-CREB-medited change in the transcriptional program, which may maintain the altered phenotype even when the cells leave the tumor environment.

Prostaglandin E2 inhibits its own renal transport by downregulation of organic anion transporters rOAT1 and rOAT3.

Prostaglandin E2 (PGE2) is the principal mediator of fever and inflammation. Recently, evidence emerged that during febrile response, PGE2 that is generated in the periphery enters the hypothalamus and contributes to the maintenance of fever. In a rat model of fever generation, peripheral PGE2 is increased, whereas clearance by metabolism of peripheral PGE2 is downregulated. The major route of PGE2 excretion is via the renal proximal tubular organic anion secretory system, where basolateral uptake that is mediated by renal organic anion transporter 1 (rOAT1) and rOAT3 is rate limiting. Therefore, it was hypothesized that PGE2 itself will abolish its excretion by rOAT1 or rOAT3. Fluorescein was used as a prototypic organic anion, and NRK-52E cells from rat served as a proximal tubular model system. PGE2 time-dependently downregulates basolateral organic anion uptake, without affecting cell volume or cell protein, recirculation of counter ions, or proximal tubular transport systems in general. In addition, PGE2 diminishes expression of both rOAT1 and rOAT3. Both organic anion uptake and expression of rOAT1 and rOAT3 are dose-dependently downregulated by PGE2. These findings suggest that during fever or inflammation, renal secretory transport of PGE2 is reduced, contributing to elevated PGE2 levels in blood. These data fit into the hypothetical concept of peripheral PGE2's playing a significant role in fever. The described regulatory mechanism may also be of relevance in chronic inflammatory events. Moreover, the data presented could explain why increased plasma urate levels occur in diseases that go along with increased levels of PGE2.

The nephrotoxin ochratoxin A induces key parameters of chronic interstitial nephropathy in renal proximal tubular cells.

Ochratoxin A (OTA) is a nephrotoxic and cancerogenic mycotoxin. There is epidemiological evidence that OTA exposition leads to cortical interstitial nephropathies in humans. However, virtually no data are available investigating the effect of OTA on renal cortical cells with respect to induction of nephropathy. Thus, we investigated whether OTA is able to induce changes of cellular properties potentially leading to interstitial nephropathy, using proximal tubular cell lines (OK, NRK-52E). OTA decreased cell number and cell protein time and dose dependently. Accordingly we investigated the effect of 100 nM or 1000 nM OTA. The decline of cell number after OTA exposure is due to necrosis and apoptosis, as measured by LDH release or DNA ladder formation and caspase-3 activation, respectively. OTA incubation of proximal tubular cells also resulted in a loss of epithelial tightness as determined by diffusion of FITC labeled inulin. Inflammation, fibrosis and epithelial-to-mesenchymal transition are described in chronic interstitial renal disease. Therefore, we also investigated the effect of OTA on NFkappaB activity, collagen secretion and generation of alpha smooth muscle actin. OTA alone was sufficient to induce the latter parameters in proximal tubular cells. Finally, OTA is a nephrotoxcic substance and elevated activity of mitogen activated protein kinases (MAPK) is described in nephropathies. As we investigated the effect of OTA on activity of ERK, JNK and p38 by ELISA, we found that OTA activates the MAPK measured dose dependently. In summary, OTA induced phenomena typical for chronic interstitial nephropathy, like loss of cells and epithelial tightness, necrosis and apoptosis as well as markers of inflammation, fibrosis and epithelial-to-mesenchymal transition in proximal tubular cells. Thus, we could show for the first time that OTA is able to induce key parameters of nephropathy in proximal tubular cells in culture. Moreover OTA interacts with MAPK and thus may exert its specific toxic actions.

Short-term regulation of basolateral organic anion uptake in proximal tubular opossum kidney cells: prostaglandin E2 acts via receptor-mediated activation of protein kinase A.

It was shown previously that EGF induces release of the important prostanoid prostaglandin E(2) (PGE(2)) in proximal tubular opossum kidney (OK) cells and PGE(2) then stimulates initial basolateral uptake of organic anions (OA) dose dependently. PGE(2) is a receptor agonist and a known substrate for the basolateral exchanger mediating OA uptake (OAT1 and/or OAT3). This study investigated the mechanism of short-term PGE(2) action on initial basolateral OA uptake in OK cells. PGE(2) stimulation of OA uptake was abolished by selective inhibition of adenylate cyclase (by MDL-12, 330A) or protein kinase A (PKA; by H89). PGE(2) stimulation of OA uptake persisted after preloading the cells with glutarate and was still abolished by inhibition of PKA. Selective activation of adenylate cyclase by forskolin led to identical results. These data contradicted the hypothesis that PGE(2) action on OA uptake is due to its action as a counter ion. Therefore, we tested whether the PGE(2) receptors (EP1 to 4) are involved in stimulation of OA uptake in OK cells by PGE(2). Because of their intracellular signaling profile, EP1 and EP3 were not taken into account as possible receptors for mediation of PGE(2)-induced OA uptake. With the use of selective agonists (11-deoxy PGE(1) and butaprost), EP4 was pharmacologically identified as the receptor responsible for PGE(2)-mediated stimulation of OA uptake. By reverse transcription-PCR, cloning, and subsequent sequencing, a homologue fragment to EP4 was identified in OK cells. EGF-induced stimulation of basolateral organic anion uptake was abolished by inhibition of adenylate cyclase or PKA. This indicates that EGF action is mediated by generation of PGE(2). The following model is proposed: PGE(2) generated in the cells does not act as a counter ion but activates adenylate cyclase. This is mediated by a homologue of EP4 receptor. cAMP then activates PKA, which stimulates initial basolateral uptake of OA in OK cells by a not-yet-known mechanism. PGE(2) is an organic anion, a potential stimulator of organic anion excretion, and an important mediator of inflammation all at once. Thus, the mechanism presented here may contribute to a limitation of inflammatory events in the kidney cortex interstitium.

Short-term regulation of basolateral organic anion uptake in proximal tubular OK cells: EGF acts via MAPK, PLA(2), and COX1.

The organic anion transport system of the kidney is of major importance for the excretion of a variety of endogenous compounds, drugs, and potentially toxic substances. The basolateral uptake into proximal tubular cells is mediated by a tertiary active transport system. Epidermal growth factor (EGF) leads to an increase in the basolateral uptake rate of the model substrate para-aminohippuric acid (PAH) in opossum kidney (OK) cells. This stimulation is mediated by successive activation of the mitogen-activated protein kinases,mitogen-activated/extracellular signal-regulated kinase kinase (MEK) and extracellular regulated kinase isoforms 1 and 2 (ERK1/2). This study investigates the regulatory network of EGF action on PAH uptake downstream ERK1/2 in more detail. EGF stimulation of the basolateral uptake rate of [(14)C]PAH was abolished by the phospholipase A(2) inhibitor AACOCF3.[(14)C]PAH uptake was enhanced by arachidonic acid. Furthermore, EGF led to an increase in arachidonic acid release and to the generation of prostaglandins. AACOCF3 did not influence EGF-induced ERK1/2 activation, indicating that ERK1/2 is upstream of PLA(2). In addition, EGF stimulated the influx of extracellular Ca(2+). However, Ca(2+)-influx was not required for the stimulatory action of EGF on [(14)C]PAH uptake. Inhibitors of COX and lipoxygenases reduced [(14)C]PAH uptake dose-dependently, whereas inhibition of cytochrome P450 did not. In the presence of indomethacin, EGF had no stimulatory effect on [(14)C]PAH uptake. The inhibitory effect of indomethacin was not due to competitive action on PAH uptake. Furthermore, prostaglandin E(2) (PGE(2)) increased basolateral [(14)C]PAH uptake rate dose-dependently, and this increase was also observed in the presence of indomethacin. Selective inhibition of COX2 by indomethacin amid or indomethacin n-heptyl ester did not inhibit [(14)C]PAH uptake, whereas selective inhibition of COX1 dose-dependently inhibited [(14)C]PAH uptake. This and previous data lead to the conclusion that EGF successively activates MEK, ERK1/2, and PLA(2), leading to an increased release of arachidonic acid. Subsequently, arachidonic acid is metabolized to prostaglandins via COX1, which then mediate EGF-induced stimulation of basolateral organic anion uptake rate.

Apical expression or expression in a non polarized cell of hOAT1 inverses regulation by epidermal growth factor (EGF) as compared to basolateral hOAT1.

Physiologically, OAT1 is located in the basolateral membrane of proximal tubular cells. During renal damage loss of polarity occurs in renal epithelial cells, leading to missorting of proteins or complete loss of polarity. Missorting or loss of polarity generally leads to disturbance of vectorial transport. In the present study, hOAT1 was expressed in human renal epithelial IHKE cells (IHKE-hOAT1) and in non polarized CHO cells (CHO-hOAT1). Because EGF and its receptor is described to play on important role in recovery from renal damage, we compared the regulation of hOAT1 by EGF in the (a) basolateral and (b) apical membrane of epithelial cells, and in (c) non polarized cells, resembling the above mentioned pathophysiological situations. Expression of hOAT1 was verified by determination of the kinetic parameters (using fluorescein as a substrate) and western blot (CHO-hOAT1) or RT-PCR (IHKE-hOAT1). To investigate the EGF effect on hOAT1, CHO-hOAT1 cells were additionally co-transfected with the human EGF receptor HER1. In agreement with previous publications, incubation of IHKE-hOAT1 cells with EGF increased fluorescein uptake via basolateral hOAT1. In opposite, EGF inhibited hOAT1 mediated fluorescein uptake across the apical membrane of IHKE-hOAT1 cells. Additionally EGF inhibited hOAT1 mediated fluorescein uptake into non polarized CHO-hOAT1-HER1 cells, too. In summary, we confirmed that EGF stimulates basolateral uptake of organic anions (a) in proximal tubular cells mediated by hOAT1. However, EGF inhibits hOAT1 located in the apical membrane (b) or in non polarized cells (c). Renal failure is associated with successive loss of epithelial polarity. Therefore, inverted regulation of hOAT1 falsely located in the apical membrane of proximal tubular cells may be part of a mechanism stabilizing organic anion secretion in pathophysiological situations.