Christoph Sauvant

Transforming growth factor‐β1 reduces megalin‐ and cubilin‐mediated endocytosis of albumin in proximal‐tubule‐derived opossum kidney cells

Transforming growth factor (TGF)‐β1 is a member of a superfamily of multifunctional cytokines involved in several pathological processes of the kidney, including fibrogenesis, apoptosis and epithelial‐mesenchymal transition. These events lead to tubulointerstitial fibrosis and glomerulosclerosis. Less is known about TGF‐β1‐induced alterations of cell function. An important function of proximal tubular cells is reabsorption of filtered proteins, including albumin, via megalin‐cubilin‐dependent receptor‐mediated endocytosis. In this study we used a well established cell culture model (proximal‐tubule‐derived opossum kidney (OK) cells) in order to test the hypothesis that TGF‐β1 reduces megalin‐cubilin‐mediated endocytosis. Previously we have shown that albumin endocytosis in OK cells is mediated by megalin/cubulin. TGF‐β1 led to a time‐ and dose‐dependent downregulation of megalin‐cubilin‐mediated endocytosis without affecting two other transport systems tested. Binding, internalization and intracellular trafficking of the ligand albumin were affected. Decreased binding resulted from reduced cubilin and megalin expression in the 200 000 g membrane fraction. The underlying mechanism of TGF‐β1 action does not involve mitogen‐activated protein kinases, protein kinase C or A, or reactive oxygen species. In contrast, TGF‐β1‐induced downregulation of megalin‐cubilin‐mediated endocytosis was sensitive to inhibition of translation and transcription and was preceded by Smad2 and 3 phosphorylation. Dominant negative Smad2/3 constructs prevented the effect of TGF‐β1. In conclusion our data indicate that enhanced levels of TGF‐β1 occurring in various nephropathies can lead to downregulation of megalin‐cubilin‐dependent endocytosis. Probably, TGF‐β1 leads to Smad2‐ and Smad3‐dependent expression of negative regulators of receptor‐mediated endocytosis.

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 (pHe, pHi) 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 pHi 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 pHe. 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 H2O2 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.

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.

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 NFκB activity, collagen secretion and generation of α 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.

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 PGE2s 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.

Short-Term Regulation of Basolateral Organic Anion Uptake in Proximal Tubular OK cells: EGF Acts via MAPK, PLA2, 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.

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 NOx-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 NOx 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.

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.

Inhibition of initial transport rate of basolateral organic anion carrier in renal PT by BK and phenylephrine.

The effect of ligands for phospholipase C-coupled receptors and of protein kinase C (PKC) stimulation with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or 1,2-dioctanoyl- sn-glycerol on the activity of the basolateral organic anion transporter (OAT) in S2 segments of single, nonperfused rabbit proximal tubules (PT) was measured with the use of fluorescein and epifluorescence microscopy. The initial uptake rate (25 s, OAT activity) was measured in real time by using conditions similar to those found in vivo. Stimulation of PKC with PMA or 1,2-dioctanoyl- sn-glycerol led to an inhibition of OAT activity, which could be prevented by 10-7 mol/l of the PKC-specific inhibitor bisindolylmaleimide. The α1-receptor agonist phenylephrine as well as the peptide hormone bradykinin induced a reversible decrease of OAT activity, which was prevented by bisindolylmaleimide. The observed effect was not due to a decrease in the concentration of the counterion α-ketoglutarate or to impaired α-ketoglutarate recycling, because it was unchanged in the continuous presence of α-ketoglutarate or methyl succinate. We conclude that physiological stimuli can inhibit the activity of OAT in rabbit PT via PKC. The effect is not mediated by alterations in counterion availability but by a direct action on the OAT.

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 (αSMA), inflammation (MCP-1), inducible NO-synthase (iNOS), release of PGE2 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.